Islet cell antigen 1851

ABSTRACT

A mammalian islet cell antigen polypeptide involved in the development of insulin-dependent diabetes mellitus (IDDM) is disclosed. This islet cell antigen polypeptide, 1851, was found to contain regions of homology to the protein tyrosine phosphatase family. Methods for diagnosis and treatment, including use in immunoprecipitation assays and the induction of immune tolerance using the recombinant mammalian polypeptides and antibodies specific to mammalian islet cell antigen 1851 polypeptides are presented.

BACKGROUND OF THE INVENTION

[0001] Insulin-dependent diabetes mellitus (IDDM) is a disease resultingfrom the autoimmune destruction of the insulin-producing β-cells of thepancreas. Studies directed at identifying the autoantigen(s) responsiblefor β-cell destruction have generated several candidates, includingpoorly characterized islet cell antigens (ICA) (Bottazzo et al., Lancet2: 1279-83, 1974), insulin (Palmer et al., Science 222: 1337-39, 1983),glutamic acid decarboxylase (GAD) (Baekkeskov et al., Nature 298:167-69, 1982; Baekkeskov et al., Nature 347: 151-56, 1990), and a 64 kDislet cell antigen that is distinct from GAD and that which yields 37 kDand 40 kD fragments upon trypsin-digestion (Christie et al., Diabetes41: 782-87, 1992).

[0002] Detection of specific autoantigens in prediabetic individuals hasbeen used as a predictive marker to identify, before clinical onset andsignificant β-cell loss has occurred, those at greater risk ofdeveloping IDDM (Gorsuch et al., Lancet 2: 1363-65, 1981; Baekkeskov etal., J. Clin. Invest. 79: 926-34, 1987; Johnstone et al., Diabetologia32: 382-86, 1989; Ziegler et al., Diabetes 38: 1320-25, 1989; Baekkeskovet al., Nature (Lond) 347: 151-56, 1990; Bonifacio et al., Lancet 335:147-49, 1990; and Bingley et al. Diabetes 43: 1304-10, 1994).

[0003] Antibodies to the 40 kD, and more particularly the 37 kD, ICAfragments are detected when clinical onset of IDDM is imminent and arefound to be closely associated with IDDM development (Christie et al.,Diabetes 41: 782-87, 1992). Diabetic sera containing antibodies specificto the 40 kD fragment were recently found to bind to the intracellulardomain of the protein tyrosine phosphatase, IA-2/ICA512 (Lu et al.,Biochem. Biophys. Res. Comm. 204: 930-36, 1994; Lan et al., DNA CellBiol. 13: 505-14, 1994; Rabin et al., J. Immunol. 152: 3183-88, 1994;Payton et al., J. Clinc. Invest. 96: 1506-11, 1995; and Passini et al.,Proc. Natl. Acad. Sci. USA 92: 9412-16, 1995). Antibodies specific tothe 37 kD fragment are thought to bind either to a posttranslational invivo modification of IA-2/ICA512 or a different, but probably related,protein precursor (Passini et al., ibid.).

[0004] ICA 512 was initially isolated as an autoantigen from an isletcell cDNA library, and was subsequently shown to be related to thereceptor-linked protein tyrosine phosphatase family (Rabin et al.,ibid.). ICA 512 was later found to be identical to a mouse and humanprotein tyrosine phosphatase, IA-2, isolated from brain and insulinomacDNA libraries (Lu et al., ibid.; and Lan et al., ibid.).

[0005] Detection of diabetes-associated autoantigens, especiallycombinations of autoantigens, genotypes, such as HLA DR and HLA DQ, andloci, such as the polymorphic region in the 5′ flanking region of theinsulin gene; in prediabetic individuals have been shown to be usefulpredictive markers of IDDM, see for example, Bell et al., (Diabetes33:176-83, 1984); Sheehy et al., (J. Clin. Invest. 83:830-35, 1989); andBingley et al., (Diabetes 43: 1304-10, 1994). There is therefore a needin the art for autoantigens that would serve to improve detection anddiagnosis of IDDM. The present invention fulfills this need by providingnovel autoantigens as well as related compositions and methods. Theautoantigens of the present invention represent a new β-cell antigen.The present invention also provides other, related advantages.

SUMMARY OF THE INVENTION

[0006] The present invention provides an isolated polynucleotide whichforms an immune complex with an autoantibody from a patient at risk ofor predisposed to develop IDDM, comprising a DNA segment encoding amammalian islet cell antigen polypeptide of SEQ ID NO:16 from Leu, aminoacid residue 636 to Gln, amino acid residue 1012. The invention alsoprovides a mammalian islet cell antigen polypeptide of SEQ ID NO:22 fromLeu, amino acid residue 442 to Gln, amino acid residue 818. Theinvention also provides allelic variants of these polypeptides. Withinone aspect of the invention, the isolated polynucleotide encodes amammalian islet cell antigen polypeptide of SEQ ID NO:22 from Phe, aminoacid residue 418, to Gln, amino acid residue 818. Within another aspectof the invention, the isolated polynucleotide encodes a mammalian isletcell antigen polypeptide of SEQ ID NO:16 from Phe, amino acid residue612, to Gln, amino acid residue 1012. The invention further providesallelic variants of these polypeptides. Within another aspect, theisolated polynucleotide encoding a polypeptide of SEQ ID NO:16 from Ala,amino acid residue 1, to Gln, amino acid residue 1012. Within anotheraspect, the isolated polynucleotide encoding a polypeptide of SEQ IDNO:22 from His, amino acid residue 1, to Gln, amino acid residue 818.The invention further provides allelic variants of these polypeptides.Within another aspect, the isolated polynucleotide is a DNA moleculecomprising a coding sequence corresponding to SEQ ID NO:21 fromnucleotide 1325 to nucleotide 2455. In still another aspect, the DNAmolecule comprises a coding sequence corresponding to SEQ ID NO:15 fromnucleotide 1909 to nucleotide 3039. The invention also provides allelicvariants of these molecules. The invention further provides complementsof polynucleotide molecules which specifically hybridize to thesemolecules. In yet another aspect, the isolated polynucleotide is a DNAmolecule comprising a coding sequence corresponding to SEQ ID NO:21 fromnucleotide 1254 to nucleotide 2455. Within another aspect, the isolatedpolynucleotide is a DNA molecule comprising a coding sequencecorresponding to SEQ ID NO:15 from nucleotide 1837 to nucleotide 3039.The invention also provides allelic variants of these molecules. Theinvention further provides complements of polynucleotide molecules whichspecifically hybridize to these molecules. In still another aspect, theDNA molecule comprises a coding sequence corresponding to SEQ ID NO:15from nucleotide 4 to nucleotide 3039. In still another aspect, the DNAmolecule comprises a coding sequence corresponding to SEQ ID NO:21 fromnucleotide 2 to nucleotide 2455. The invention also provides allelicvariants of these molecules. The invention further provides complementsof polynucleotide molecules which specifically hybridize to thesemolecules. The invention also provides an isolated polynucleotidemolecule which encodes a complete coding sequence of a mammalian isletcell antigen polypeptide comprising the sequence of SEQ ID NO:22 fromLeu, amino acid residue 442 to Arg, amino acid residue 738. Theinvention also provides mammalian islet cell antigens that are primateislet cell antigens.

[0007] The invention also provides DNA constructs comprising a first DNAsegment encoding a human islet cell antigen polypeptide operably linkedto additional DNA segments required for the expression of the first DNAsegment. The invention further provides a first DNA segment that is anisolated polynucleotide molecule encoding a human islet cell antigenpolypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu,amino acid residue 442 to Gln, amino acid residue 818. The inventionalso provides a first DNA segment that is an isolated polynucleotidemolecule encoding a human islet cell antigen polypeptide comprising theamino acid sequence of SEQ ID NO:22 from Leu, amino acid residue 442 toGln, amino acid residue 818. Within another aspect, the inventionprovides a first DNA segment that is an isolated polynucleotide moleculeencoding a human islet cell antigen polypeptide comprising the aminoacid sequence of SEQ ID NO:22 from His, amino acid residue 1, to Gln,amino acid residue 818. The invention further provides host cellscontaining such DNA constructs, as well as methods for producing humanislet cell antigen polypeptides comprising the steps of culturing suchhost cell and isolating the human islet cell antigen polypeptide.

[0008] The invention further provides isolated mammalian islet cellantigen polypeptides, wherein said isolated mammalian islet cell antigenpolypeptide forms an immune complex with an autoantibody from a patientat risk of or predisposed to develop IDDM comprising the amino acidsequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, aminoacid residue 818. The invention further provides isolated mammalianislet cell antigen polypeptides comprising the amino acid sequence ofSEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue1012.

[0009] The invention also provides isolated polypeptides of SEQ ID NO:16from Phe, amino acid residue 612 to Gln, amino acid residue 1012. Theinvention also provides isolated polypeptides of SEQ ID NO:22 from Phe,amino acid residue 418, to Gln, amino acid residue 818. The inventionfurther provides isolated polypeptides of SEQ ID NO:16 from Ala, aminoacid residue 1 to Gln, amino acid residue 1012. The invention alsoprovides isolated polypeptides of SEQ ID NO:22 from His, amino acidresidue 1, to Gln, amino acid residue 818. The invention furtherprovides allelic variants of these polypeptides. The invention stillfurther provides an isolated polypeptide which is a full lengthmammalian islet cell antigen protein comprising the sequence of SEQ IDNO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.The invention also provides mammalian islet cell antigens that areprimate islet cell antigens.

[0010] Within yet another aspect of the invention is provided a methodfor determining the presence of an autoantibody to a human islet cellantigen polypeptide in a biological sample, comprising the steps ofcontacting the biological sample with the human islet cell antigenpolypeptide, which comprises an amino acid sequence selected from thegroup consisting of a polypeptide of SEQ ID NO:22 from Leu, amino acidresidue 442 to Gln, amino acid residue 81, a polypeptide of SEQ ID NO:22from Phe, amino acid residue 418 to Gln, amino acid residue 818, apolypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, aminoacid residue 818, and allelic variants thereof, under conditionsconducive to immune complex formation, and detecting the presence ofimmune complex formation between the human islet cell antigenpolypeptide and the autoantibody to a human islet cell antigen, therebydetermining the presence of autoantibodies to the human islet cellantigen in the biological sample. The invention further provides humanislet cell antigen polypeptides that are detectably labeled.

[0011] Within a further embodiment the invention provides a method forpredicting the clinical course of diabetes in a patient, comprisingtesting a biological sample from a patient for the presence of humanislet cell antigen polypeptides comprising the amino acid sequenceselected from the group consisting of a polypeptide of SEQ ID NO:22 fromLeu, amino acid residue 442 to Gln, amino acid residue 81, a polypeptideof SEQ ID NO:22 from Phe, amino acid residue 418 to Gln, amino acidresidue 818, a polypeptide of SEQ ID NO:22 from His, amino acid residue1 to Gln, amino acid residue 818, and allelic variants thereof, whereinthe polypeptide forms an immune complex with an autoantibody from apatient at risk of or predisposed to develop IDDM, and classifying thepatient for clinical course of diabetes based on the presence or absenceof human islet cell antigens in the sample. The invention furtherprovides a method of predicting the clinical course of IDDM by testingone or more additional predictive markers associated with risk of orprotection from IDDM. The invention provides methods of predicting theclinical course where the predictive marker is an autoantibody to anantigen selected from the group consisting of GAD65, IA-2/ICA512 orinsulin. The invention also provides methods wherein the predictivemarker is a genotype selected from the group consisting of HLA DR andHLA DQ. The invention also provides methods wherein the predictivemarker is a polymorphic region in the 5′ flanking region of a humaninsulin gene.

[0012] The invention also provides a method for treating a patient toprevent an autoimmune response to a human islet cell antigen polypeptidecomprising inducing immunological tolerance in the patient byadministering a mammalian islet cell antigen polypeptide comprising theamino acid sequence selected from the group consisting of a polypeptideof SEQ ID NO:22 from Leu, amino acid residue 442 to Gln, amino acidresidue 81, a polypeptide of SEQ ID NO:22 from Phe, amino acid residue418 to Gln, amino acid residue 818, a polypeptide of SEQ ID NO:22 fromHis, amino acid residue 1 to Gln, amino acid residue 818, and allelicvariants thereof, that specifically binds a human islet cell antigenreceptor on immature or mature T or B lymphocytes.

[0013] The invention also provides oligonucleotide probes of at leastabout 16 nucleotides, wherein which the oligonucleotide is at least 85%homologous to a sequence of the mammalian islet cell antigen DNAsequence of SEQ ID Nos:15 or 21.

[0014] The invention further provides isolated antibodies whichspecifically bind to human islet cell antigen polypeptides whichcomprise the amino acid sequence selected from the group consisting of apolypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, aminoacid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ IDNO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, andallelic variants thereof. Within another aspect, the invention providesmonoclonal antibodies. Within yet another aspect, the invention providesa hybridoma which produces the monoclonal antibody.

[0015] The invention also provides a diagnostic kit for use in detectingautoantibodies to pancreatic β-islet cells, comprising a containercontaining an islet cell antigen polypeptide comprising an amino acidsequence selected from the group consisting of a polypeptide of SEQ IDNO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 81, apolypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln,amino acid residue 818, a polypeptide of SEQ ID NO:22 from His, aminoacid residue 1 to Gln, amino acid residue 818, and allelic variantsthereof, wherein the polypeptide forms an immune complex withautoantibodies from a patient at risk of or predisposed to develop IDDM,and one or more containers containing additional reagents.

[0016] Within another embodiment of the invention is provided apharmaceutical composition comprising an islet cell antigen comprisingan amino acid sequence selected from the group consisting of apolypeptide of SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 81, a polypeptide of SEQ ID NO:22 from Phe, aminoacid residue 418 to Gln, amino acid residue 818, a polypeptide of SEQ IDNO:22 from His, amino acid residue 1 to Gln, amino acid residue 818, andallelic variants thereof, in combination with a pharamceuticallyacceptable carrier or vehicle.

[0017] Within a further embodiment of the invention is provided a methodfor monitoring the disease state in a patient comprising testing abiological sample from a patient for the presence of human islet cellantigen post-translationally modified polypeptides, determining theconcentration of the peptides and correlating the peptide levels in thesample with the disease state in the patient. The invention providesthat the human islet cell antigen post-translationally modifiedpolypeptide comprises the sequence of SEQ ID NO:22 from His, amino acidresidue 1 to Glu, amino acid residue 227. The invention further providesthat the biological sample is plasma or serum.

[0018] Within yet a further embodiment, the invention provides a methodfor monitoring the disease state in a patient comprising exposing Tcells to islet cell antigen 1851 peptides, detecting T and correlating Tcell reactivity with disease state. The invention provides that the Tcells are from peripheral blood mononuclear cells from a prediabeticpatient. The invention further provides that the disease state isconversion from prediabetes to diabetes.

DETAILED DESCRIPTION OF THE INVENTION

[0019] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter:

[0020] Allelic variant—Any of two or more alternative forms of a geneoccupying the same chromosomal locus. Allelic variation arises naturallythrough mutation, and may result in phenotypic polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequence. The term allelic variant is also used herein to denote aprotein encoded by an allelic variant of a gene.

[0021] Biological sample—A sample that is derived from or containscells, cell components or cell products, including, but not limited to,cell culture supernatants, cell lysates, cleared cell lysates, cellextracts, tissue extracts, blood plasma, serum, and fractions thereof,from a patient.

[0022] Complements of polynucleotide molecules—Polynucleotide moleculeshaving a complementary base sequence and reverse orientation as comparedto a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ iscomplementary to 5′ CCCGTGCAT 3′.

[0023] Immune Complex Formation—A noncovalently bound molecule formedbetween an antigen and an antibody specific for that antigen, resultingin an extensively cross-linked mass. Conditions conducive to complexformation are known in the art and easily adaptable by those skilled inart, for example, the degree of complex formation is in proportion tothe relative amounts of available antigen and antibody. Such complexescan be used, for example, to identify and/or quantify the presence ofeither antigen or antibody in a biological sample, identify andcharacterize particular antibodies in tissues and cells, or to stimulatean immune response.

[0024] Isolated—When applied to a protein the term “isolated” indicatesthat the protein is found in a condition other than its nativeenvironment, such as apart from blood and animal tissue. In a preferredform, the isolated protein is substantially free of other proteins,particularly other proteins of animal origin. It is preferred to providethe proteins in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When applied to a polynucleotidemolecule the term “isolated” indicates that the molecule is removed fromits natural genetic milieu and is thus free of other extraneous orunwanted coding sequences, and is in a form suitable for use withingenetically engineered protein production systems. Such isolatedmolecules are those that are separated from their natural environmentand include cDNA and genomic clones. Isolated DNA molecules of thepresent invention are free of other genes with which they are ordinarilyassociated and may include naturally occurring 5′ and 3′ untranslatedregions such as promoters and terminators, the identification of suchwill be evident to one of ordinary skill in the art (see for example,Dynan and Tijan, Nature 316: 774-78, 1985).

[0025] Operably linked—Indicates that the segments are arranged so thatthey function in concert for their intended purposes, e.g.,transcription initiates in the promoter and proceeds through the codingsegment to the terminator.

[0026] The DNA sequences encoding the polypeptides of the presentinvention were unexpectedly identified during screening of a primateislet cell cDNA library, and human insulinoma cDNA, for autoantigenstoward human diabetic sera. Analysis of the macaque cDNA clones revealeda unique, previously unknown islet cell antigen which contained regionsof homology to the protein tyrosine phosphatase family, especially theprotein tyrosine phosphatase IA2/ICA512. This novel islet cell antigenhas been designated 1851 or ICA512β.

[0027] The present invention provides islet cell antigen polypeptideswhich are β-cell autoantigens. These autoantigens were reactive withhuman prediabetic and diabetic sera. The invention also provides methodsfor using the islet cell antigen polypeptides for the detection,diagnosis, and treatment of IDDM.

[0028] Representative islet cell antigen polypeptides of the presentinvention comprise the amino acid sequences in SEQ ID NOs:4, 16 or 22and/or are encoded by polynucleotide sequences comprising the sequencesof SEQ ID NOs:3, 15 and 21 and form an immune complex withautoantibodies from a patient at risk of or predisposed to develop IDDM.The islet cell antigen polypeptides of the present invention arepreferably from mammals, especially primates including humans. Preferredpolypeptides of the present invention include isolated polypeptidesselected from the group consisting of a polypeptide of SEQ ID NO:2 fromLeu, amino acid residue 265, to Gln amino acid residue 641. Theinvention also provides polypeptides of SEQ ID NO:2 from Glu, amino acidresidue 1, to Gln, amino acid residue 641. The invention furtherprovides macaque polypeptides of SEQ ID NO:16 from Ala, amino acidresidue 1 to Gln, amino acid residue 1012 and human polypeptides of SEQID NO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818and SEQ ID NO:22 from His, amino acid residue 1 to Gln, amino acidresidue 818. The invention further provides allelic variants andisolated sequences that are substantially identical to therepresentative polypeptide sequences of SEQ ID NOs:2, 16 and 22 andtheir species homologs. The term “substantially identical” is usedherein to denote proteins having 50%, preferably 60%, more preferably70%, and most preferably at least 80%, sequence identity to therepresentative sequences shown in SEQ ID NO:2, 16 or 22 or its specieshomologs. Within preferred embodiments, such proteins will be at least90% identical, and most preferably 95% or more identical, to SEQ IDNO:2, 16 or 22 or their species homologs.

[0029] Percent sequence identity is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986;Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 1 (amino acids are indicated by the standard one-lettercodes). The percent identity of the optimum alignment is then calculatedas: TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6D −2 2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −2 0 0 2 −4 2 5 G 0 −2 0 −1−3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1−2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2−3 −1 0 −2 −3 −2 1 −2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1−2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 2 0 −1 0 0 0 −1 −2 −2 0−1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −2 −1 −1 −1 −2 −1 1 5 W −3 −3 −4−4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −2 −2 −3 2−1 −1 −2 −2 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −2 −2 −2 −3 −3 3 2 −2 1 −1 −2 −20 −3 −1 4

[0030]$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{\quad {{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

[0031] Substantially identical proteins are characterized as having oneor more amino acid substitutions, deletions or additions. These changesare preferably of a minor nature, that is conservative amino acidsubstitutions (see Table 2) and other substitutions that do notsignificantly affect the folding or activity of the protein; smalldeletions, typically of one to about 30 amino acids; amidation of theamino- or carboxyl-terminal; and small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue, a small linkerpeptide of up to about 20-25 residues, or a small extension thatfacilitates purification, such as a poly-histidine tract, an antigenicepitope or a binding domain. See, in general, Ford et al., ProteinExpression and Purification 2: 95-107, 1991, which is incorporatedherein by reference. TABLE 2 Conservative amino acid substitutionsBasic: arginine lysine histidine Acidic: glutamic acid aspartic acidPolar: glutamine asparagine Hydrophobic: leucine isoleucine valineAromatic: phenylalanine tryptophan tyrosine Small: glycine alanineserine threonine methionine

[0032] Essential amino acids in the polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244, 1081-85, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g. protein tyrosine phosphatase activity, Strueliet al., EMBO J. 9: 2399-407, 1990, or binding to autoantibodies inprediabetic or diabetic sera) to identify amino acid residues that arecritical to the activity of the molecule. Sites of ligand-receptorinteraction can also be determined by analysis of crystal structure asdetermined by such techniques as nuclear magnetic resonance,crystallography or photoaffinity labeling. See, for example, de Vos etal., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.

[0033] Multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina protein, selecting for functional protein, and then sequencing themutagenized proteins to determine the spectrum of allowablesubstitutions at each position. These methods allow the rapiddetermination of the importance of individual amino acid residues in aprotein of interest, and can be applied to proteins of unknownstructure.

[0034] The present invention further provides isolated polynucleotidemolecules encoding islet cell antigen polypeptides which form immunecomplexes with autoantibodies from a patient at risk of or predisposedto develop IDDM. Useful polynucleotide molecules in this regard includemRNA, genomic DNA, cDNA and synthetic DNA. For production of recombinantislet cell antigen polypeptides, cDNA is preferred. The inventionprovides an isolated polynucleotide molecule wherein the molecule is aDNA molecule comprising a coding sequence corresponding to SEQ ID NO:1from nucleotide 795 to nucleotide 1922. The invention also provides aDNA molecule comprising a coding sequence corresponding to SEQ ID NO:1from nucleotide 1 to nucleotide 2168. The invention also provides a DNAmolecule comprising a coding sequence corresponding to nucleotide 4 tonucleotide 3039 of SEQ ID NO: 15. The invention also provides DNAmolecules from nucleotide 1325 to nucleotide 2455, from nucleotide 1254to nucleotide 2455 and from nucleotide 2 to nucleotide 2544 of SEQ IDNO:21. The invention also provides allelic variants of the sequencesshown in SEQ ID NOs:1, 15 or 21, and polynucleotide molecules thatspecifically hybridize to allelic variants. Such polynucleotidemolecules will hybridize to the representative DNA sequences of SEQ IDNOs:1, 15, 21 or their allelic variants under stringent conditions(Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y., 1989). As used herein, the term“stringent conditions” refers to hybridizing conditions that employ lowionic strength and high temperature for washing, for example, 0.015 MNaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.; employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% polyvinylpyrrolidone/50 mM sodium citrateat 42° C.; or employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075M sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS. Such hybridizable polynucleotide moleculeswould include genetically engineered or synthetic variants of therepresentative islet cell antigen polynucleotide sequence, SEQ ID NO: 1,and polynucleotide molecules that encode one or more amino acidsubstitutions, deletions or additions, preferably of a minor nature, asdiscussed above. Genetically engineered variants may be obtained byusing oligonucleotide-directed site-specific mutagenesis, by use ofrestriction endonuclease digestion and adapter ligation, polymerasechain reaction (PCR), or other methods well established in theliterature (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989, andSmith et al., Genetic Engineering: Principles and Methods, Plenum Press,1981; which are incorporated herein by reference). In addition,hybridizable polynucleotide molecules may encompass sequences containingdegeneracies in the DNA code wherein host-preferred codons aresubstituted for the analogous codons in the representative sequences ofSEQ ID NOs: 1, 15 and 21.

[0035] Analysis of the representative cDNA sequences of SEQ ID NO:1, 15and 21 and their representative polypeptide sequences of SEQ ID NO:2, 16and 22, show that they contain regions of homology to transmembraneprotein tyrosine phosphatases. Comparison of the human protein tyrosinephosphatase IA-2/ICA512 cDNA and amino acid sequences with those of 1851suggests that the coding region of macaque 1851 is missingamino-terminal sequence corresponding to approximately 1 amino acid andhuman 1851 is missing approximately 200 amino acid residues of the aminoterminus. To recover the 5′ region, cDNA libraries from differenttissues can be screened to obtain a full length cDNA, which encodes afull length mammalian islet cell antigen polypeptides. Another optionfor obtaining the complete coding sequence comprises using 5′ RACE(Rapid Amplification cDNA Ends) PCR. RACE is an art recognized PCR-basedmethod for amplifying the 5′ ends of incomplete cDNAs, a frequentoccurrence in cDNA cloning. To obtain the 5′ portion of a cDNA, PCR iscarried out on specially prepared cDNA which contains unique anchorsequences, using anchor primers provided with the 5′ RACE reagentsavailable from, for example, Clontech, Palo Alto, Calif. and a 3′ primerbased on known sequence. The 5′-RACE-Ready cDNA can be purchasedcommercially (Clontech), or prepared according to known methods. Asecondary PCR reaction can then be carried out using the anchor primerand a nested 3′ primer, according to known methods. Once a full-lengthcDNA is obtained, it is expressed and analyzed for overall structuralsimilarity to known protein tyrosine phosphatases, and examined forfeatures such as a continuous open reading frame flanked by translationinitiation and termination sites and a potential signal sequence.

[0036] Transmembrane, or receptor-linked, protein tyrosine phosphatasesconsist of a conserved cytoplasmic domain which may have one or two(tandemly duplicated) catalytic regions, a single transmembrane domain,a highly variable extracellular domain and a signal peptide. Thesestructural features suggest that receptor-linked protein tyrosinephosphatases would be capable of binding ligand and transducing externalsignal, but no ligands as of yet have been identified. Based on therepresentative amino acid sequence of SEQ ID NOs:2 and 15, the macaque1851 polypeptide has an approximately 611 amino acid extracellulardomain, from Ala, amino acid residue 1 to Lys, amino acid residue 611 ofSEQ ID NO:16, containing a post translational modification dibasic site,at amino acid residue 423-424, or a tribasic site at amino acid residues422-424; a 24 amino acid transmembrane domain comprising amino acidresidue 241 to amino acid residue 265 of SEQ ID NO:2 or Phe, amino acidresidue 612 to Cys, amino acid residue 635 of SEQ ID NO:16 and anapproximately 375 amino acid cytoplasmic domain comprising the aminoacid residue 265 to amino acid residue 640 of SEQ ID NO:2 or Leu, aminoacid residue 636 to Gln, amino acid residue 1012 of SEQ ID NO:16. Therepresentative amino acid sequence of the human islet cell antigen 1851(SEQ ID NO:22) has 417 amino acids of an extracellular domain, from His,amino acid residue 1 to Lys, amino acid residue 417 of SEQ ID NO:22; a24 amino acid residue transmembrane domain, from Phe, amino acid residue418 to Cys, amino acid residue 441, of SEQ ID NO:22; and a 376 aminoacid cytoplasmic domain, from Leu, amino acid residue 442 to Gln, aminoacid residue 818 of SEQ ID NO:22.

[0037] The cytoplasmic domain of 1851 contains many regions that areconserved between members of the protein tyrosine phosphatase family.Within the cytoplasmic domain of protein tyrosine phosphatases is acatalytic region of about 230 amino acids, which contains a highlyconserved catalytic core segment of approximately 11 amino acid residues(VHCXAGXXRXG SEQ ID NO:13) where the first three X's are any amino acid,the fourth X is S or T, and the cysteine appears to be essential to thecatalytic mechanism (Fischer et al., Science 253: 401-06). The catalyticcore sequence of the representative macaque 1851 polypeptide sequencesof SEQ ID Nos:2 and 16 and human 1851 polypeptide sequence representedby SEQ ID NO:22 differs from other members of the protein tyrosinephosphatase family in that alanine has been replaced by aspartic acidand the second variable amino acid (X) is alanine. 1851, likeIA-2/ICA512, has a single catalytic region. Deletion of C-terminal aminoacids from the intracellular domain of human islet cell antigen 1851reduced reactivity with new onset IDDM sera, suggesting this region mayplay a role in defining an autoantibody epitope. Removal of theC-terminal 27 amino acids decreased reactivity from 19/53 sera (36%) to10/53 sera (19%), a 47% decrease. Removal of the C-terminal 80 aminoacids decreased reactivity further to 9/53 sera (17%), a 53% decrease,and removal of the C-terminal 160 amino acids abolished all recognitionby all 53 new onset IDDM sera. This is similar to the reports of one oftwo described intracellular IA-2/ICA512 autoantibody epitopes (Bonifacioet al., J. Immunol. 155:5419-426, 1995). That human islet cell antigens1851 and human IA-2/ICA512 are each precipitated by sera that do notprecipitate the other suggests that each antigen has unique autoantibodyepitopes, which is consistent with previous findings regarding the 37 kDand 40 kD tryptic fragments (Payton et al., J. Clin. Invest. 96:1506-11,1995). A comparison between the overall human and macaque islet cellantigen 1851 nucleotide and amino acid sequences shows a 96.2%nucleotide identity and a 94.6% amino acid identity, in particular therewas 97% identity within the nucleotide sequence and 98.9% identitywithin the amino acid sequence of the corresponding cytoplasmic domains,100% identity within the transmembrane domain. There is 77% amino acididentity within the cytoplasmic domain between the claimed human (SEQ IDNO:22) and macaque (SEQ ID NO:16) islet cell antigen 1851 sequences andthe reported human IA-2/ICA512 sequences (Lan et al., ibid.; and Rabinet al., ibid.). Between the full length macaque islet cell antigen 1851sequence (as represented in SEQ ID Nos:15 and 16) and rat phogrinsequences (Wasmeier and Hutton, J. Biol. Chem. 271:18161-70, 1996) therewas less homology, 75.5% identity within the nucleotide sequence and69.9% identity within the amino acid sequence.

[0038] In contrast, there is little homology in the extracellularregions of transmembrane protein tyrosine phosphatases. Some containIg-like and/or fibronectin type III repeats (Streuli et al., J. Exp.Med. 168: 1523, 1988; Hariharan et al., Proc. Natl. Acad. Aci. USA 88:11266, 1991); others have glycosylated segments (Sap et al., Proc. Natl.Acad. Sci. USA 87:6112, 1990; and Krueger et al., EMBO J. 9: 3241, 1990)and a conserved cysteine-rich region (Tonks et al., J. Biol. Chem. 265:10674-80, 1990) (Lan et al. ibid.). There is 31% identity betweenmacaque islet cell antigen 1851 (as represented by SEQ ID NO:15) andIA-2/ICA512 (Lan et al., ibid.; and Rabin et al., ibid.) within theextracellular domain.

[0039] The tissue distribution of human islet cell antigen 1851 isgenerally neuroendocrine. Northern analysis showed strong hybridizationto human mRNA from brain and pancreas and weaker hybridization in spinalcord, thyroid, adrenal and GI tract. In situ hybridization using macaquetissues further localized pancreatic and adrenal expression to isletsand adrenal medulla, respectively. Northern blot analysis of rat phogrinshowed expression in brain, pancreas and α and βcell tumor lines(Wasmeier and Hutton, ibid.); mouse IA-2β in brain, pancreas, stomachand in insulinoma and glucagomoma cell lines (Lu et al., Proc. Natl.Acad. Sci. USA 93:2307-11, 1996); human IA-2 in brain, pituitary andpancreas, four insulinoma cell lines and a glioblastoma cell line (Lanet al., ibid.); and human ICA512, brain and pancreas (Rabin et al.,ibid.).

[0040] Limited trypsinization of IA-2/ICA512 and human islet cellantigen 1851 yielded a 40 kD IA-2/ICA512 fragment and a 37 kD islet cellantigen 1851 fragment. These correspond to the 37 kD and 40 kD trypticfragments described by Christie et al. (J. Exp. Med. 172:789-94, 1990),Payton et al. (J. Clin. Invest. 96:1506-11, 1995), Bonifacio et al. (J.Immunol. 155:5419-26, 1995), Lu et al. (Proc. Natl. Acad. Sci. USA93:2307-11, 1996) and Wasmeier and Hutton (ibid.).

[0041] Members of the protein tyrosine phosphatase family have beenshown to display alternative mRNA splicing (Moeller et al., WO 94/21800;Hall et al., J. Immunol. 141: 2781-87, 1988; Johnson et al., J. Biol.Chem. 264: 6220-29, 1989; Streuli and Saito, EMBO J. 8: 787-96, 1989;Matthews et al., Proc. Natl. Acad. Sci. USA 87: 4444-48, 1990; Waltonand Dixon, Ann. Rev. Biochem. 62: 101-20, 1993; and Pan et al., J. Biol.Chem. 268: 19284-91, 1993). Alternative splicing may be important inautoantibody recognition; “inappropriate” splicing could lead toautoimmunity by activating T cells, for example.

[0042] The invention provides isolated DNA molecules that are useful inproducing recombinant islet cell antigens. As will be evident to oneskilled in the art, each individual domain or combinations of thedomains may be prepared synthetically or by recombinant DNA techniquesfor use in the present invention. Thus, the present invention providesthe advantage that islet cell antigens are produced in high quantitiesthat may be readily purified using methods known in the art (seegenerally; Scopes, Protein Purification, Springer-Verlag, NY, 1982).Alternatively, the proteins of the present invention may be synthesizedfollowing conventional synthesis methods, such as the solid-phasesynthesis method of Barany and Merrifield (in The Peptides. Analysis,Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press,NY, pp. 1-284, 1980), by partial solid-phase techniques, by fragmentcondensation or by classical solution addition.

[0043] DNA molecules of the present invention can be isolated usingstandard cloning methods such as those described by Maniatis et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982;which is incorporated herein by reference), Sambrook et al., (MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,1989), or Mullis et al. (U.S. Pat. No. 4,683,195) which are incorporatedherein by reference. Alternatively, the coding sequences of the presentinvention can be synthesized using standard techniques that are wellknown in the art, such as by synthesis on an automated DNA synthesizer.

[0044] The sequence of a polynucleotide molecule encoding arepresentative islet cell antigen polypeptide is shown in SEQ ID NOs: 1,15 and 21 and the corresponding amino acid sequences are shown in SEQ IDNOs: 2, 16 and 22. Those skilled in the art will recognize that thesesequences correspond to one allele of either the macaque or human gene,and that allelic variation is expected to exist. Allelic variants of theDNA sequence shown in SEQ ID NO: 1, 15 and 21 including those containingsilent mutations and those in which mutations result in amino acidsequence changes, are within the scope of the present invention, as areproteins which are allelic variants of SEQ ID NO: 2, 16 and 22.

[0045] The macaque sequence disclosed herein is useful for isolatingpolynucleotide molecules encoding islet cell antigen polypeptides fromother species (“species homologs”). In particular, the macaque cDNA wasused to conduct a sequence search for a human homolog. A match was foundas an expressed sequence tag (EST) from a human fetal brain librarysubmitted to the Genbank database (GenBank ID: TO361, clone ID:HFBCV88). This 127 amino acid polypeptide, SEQ ID NO:5, had homology toa region of the cytoplasmic domain of M1.18.5.1 (SEQ ID NO:2) and wasused to design PCR primers to clone a 1.1 kD cytoplasmic portion (SEQ IDNOs:6 and 7) of the human 1851 sequence, as described in the examplesbelow. Other preferred species homologs include mammalian homologs suchas bovine, canine, porcine, ovine, and equine proteins. Methods forusing sequence information from a first species to clone a correspondingpolynucleotide sequence from a second species are well known in the art.See, for example, Ausubel et al., eds., Current Protocols in MolecularBiology, John Wiley and Sons, Inc., NY, 1987.

[0046] DNA molecules of the present invention or portions thereof may beused as probes, for example, to directly detect 1851 sequences in cellsor biological samples. Such DNA molecules are generally syntheticoligonucleotides, but may be generated from cloned cDNA or genomicsequences and will generally comprise at least about 16 nucleotides,more often from about 17 nucleotides to about 25 or more nucleotides,sometimes 40 to 60 nucleotides, and in some instances a substantialportion or even the entire 1851 gene or cDNA. The syntheticoligonucleotides of the present invention have at least 85% identity toa representative macaque or human 1851 DNA sequence (SEQ ID Nos:1, 15and 21) or their complements. For use as probes, the molecules arelabeled to provide a detectable signal, such as with an enzyme, biotin,a radionuclide, fluorophore, chemiluminescer, paramagnetic particle,etc., according to methods known in the art. Probes of the presentinvention may also be used in diagnostic methods to detectautoantibodies in diabetic and prediabetic sera.

[0047] DNA molecules used within the present invention may be labeledand used in a hybridization procedure similar to the Southern or dotblot. As will be understood by those skilled in the art, conditions thatallow the DNA molecules of the present invention to hybridize to therepresentative DNA sequence of SEQ ID NO:1, 15 or 21 or their allelicvariants may be determined by methods well known in the art (reviewed,for example, by Sambrook et al. Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., 1989; which is incorporatedherein by reference). Those skilled in the art will be capable ofvarying hybridization conditions (i.e. stringency of hybridization) ofthe DNA molecules as appropriate for use in the various procedures bymethods well known in the literature (see, for example, Sambrook et al.,ibid., pages 11.45-11.53). The higher the stringency of hybridization,the lower the number of mismatched sequences detected. Alternatively,lower stringency will allow related sequences to be identified.

[0048] Alternatively, allelic variants may be identified using DNAmolecules of the present invention and, for example, the polymerasechain reaction (PCR) (disclosed by Saiki et al., Science 239: 487, 1987;Mullis et al., U.S. Pat. No. 4,686,195; and Mullis et al., U.S. Pat. No.4,683,202) to amplify DNA sequences, which are subsequently detected bytheir characteristic size on agarose gels or which may be sequenced todetect sequence abnormalities.

[0049] DNA molecules encoding the islet cell antigen polypeptides of thepresent invention may be inserted into DNA constructs. As used withinthe context of the present invention a DNA construct is understood torefer to a DNA molecule, or a clone of such a molecule, either single-or double-stranded, which has been modified through human interventionto contain segments of DNA combined and juxtaposed in a manner thatwould not otherwise exist in nature. DNA constructs of the presentinvention comprise a first DNA segment encoding an islet cell antigenpolypeptide operably linked to additional DNA segments required for theexpression of the first DNA segment. Within the context of the presentinvention, additional DNA segments will generally include promoters andtranscription terminators, and may further include enhancers and otherelements. One or more selectable markers may also be included. DNAconstructs useful for expressing cloned DNA segments in a variety ofprokaryotic and eukaryotic host cells can be prepared from readilyavailable components or purchase from commercial suppliers.

[0050] In general, a DNA sequence encoding a protein of the presentinvention is operably linked to a transcription promoter and terminatorwithin a DNA construct. The construct will commonly contain one or moreselectable markers and one or more origins of replication, althoughthose skilled in the art will recognize that within certain systemsselectable markers may be provided on separate vectors, and replicationof the exogenous DNA may be provided by integration into the host cellgenome. Selection of promoters, terminators, selectable markers, vectorsand other elements is a matter of routine design within the level ofordinary skill in the art. Many such elements are described in theliterature and are available through commercial suppliers.

[0051] In one embodiment the first DNA segment is an isolatedpolynucleotide molecule encoding a mammalian islet cell antigenpolypeptide comprising the amino acid sequence of SEQ ID NO:4, whereinthe polypeptide forms an immune complex with autoantibodies from apatient at risk of or predisposed to IDDM. In another embodiment, thefirst DNA segment is an isolated polynucleotide encoding a polypeptideof SEQ ID NO:2 from Leu, amino acid residue 265 to Gln, amino acidresidue 641. In another embodiment, the first DNA segment is an isolatedpolynucleotide encoding a polypeptide of SEQ ID NO:2 from Ser, aminoacid residue 1, to Gln, amino acid residue 641.

[0052] Within yet another embodiment, the first DNA segment is anisolated polynucleotide encoding a polypeptide of SEQ ID NO:22 from Leu,amino acid residue 442 to Gln, amino acid residue 818. In anotherembodiment, the first DNA segment is an isolated polynucleotide encodinga polypeptide of SEQ ID NO:16 from Ala, amino acid residue 1 to Gln,amino acid residue 1012.

[0053] The proteins of the present invention can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Techniquesfor manipulating cloned DNA molecules and introducing exogenous DNA intoa variety of host cells are disclosed by Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., ibid., whichare incorporated herein by reference.

[0054] To direct a protein of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) is provided in theexpression vector. The secretory signal sequence is joined to the DNAsequence encoding a protein of the present invention in the correctreading frame. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the protein of interest, although certainsignal sequences may be positioned elsewhere in the DNA sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland etal., U.S. Pat. No. 5,143,830). The secretory signal sequence may be thatnormally associated with a protein of the present invention, or may befrom a gene encoding another secreted protein.

[0055] Cultured mammalian cells are also preferred hosts within thepresent invention. A preferred vector system for use in the presentinvention is the pZCEP vector system as disclosed by Jelineck et al.,Science, 259: 1615-16, 1993. Methods for introducing exogenous DNA intomammalian host cells include calcium phosphate-mediated transfection(Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic CellGenetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973),electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextranmediated transfection (Ausubel et al., eds., Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, 1987), and cationiclipid transfection using commercially available reagents including theBoehringer Mannheim Transfection-Reagent(N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl ammoniummethylsulfate;Boehringer Mannheim, Indianapolis, Ind.) or LIPOFECTIN˜ reagent(N-[1-(2,3-Dioleyloxy)propyl]-N,N,N-trimethylammonium chloride anddioeleoyl phosphatidylethanolamine; GIBCO-BRL, Gaithersburg, Md.) usingthe manufacturer-supplied directions, which are incorporated herein byreference. The production of recombinant proteins in cultured mammaliancells is disclosed, for example, by Levinson et al., U.S. Pat. No.4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S.Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134, which areincorporated herein by reference. Preferred cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus.

[0056] Prokaryotic cells can also serve as host cells for use incarrying out the present invention. Particularly preferred are strainsof the bacteria Escherichia coli, although Bacillus and other genera arealso useful. Techniques for transforming these hosts and expressingforeign DNA sequences cloned therein are well known in the art (see,e.g., Sambrook et al., ibid.). When expressing the proteins in bacteriasuch as E. coli, the protein may be retained in the cytoplasm, typicallyas insoluble granules, or may be directed to the periplasmic space. Inthe former case, the cells are lysed, and the granules are recovered anddenatured using, for example, guanidine isothiocyanate. The denaturedprotein is then refolded by diluting the denaturant. In the latter case,the protein can be recovered from the periplasmic space in a solubleform.

[0057] Fungal cells are also suitable as host cells. For example,Saccharomyces ssp., Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia guillermondii, Pichia methanolica, and Candida maltosatransformation systems are known in the art. See, for example, Kawasaki,U.S. Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373,Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743;and Murray et al., U.S. Pat. No. 4,845,075, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349, which is incorporated herein byreference. Methods for transforming Acremonium chrysogenum are disclosedby Sumino et al., U.S. Pat. No. 5,162,228, which is incorporated hereinby reference.

[0058] Other higher eukaryotic cells can also be used as hosts,including insect cells, plant cells and avian cells. Transformation ofinsect cells and production of foreign proteins therein is disclosed byGuarino et al., U.S. Pat. No. 5,162,222 and Bang et al., U.S. Pat. No.4,775,624, which are incorporated herein by reference. The use ofAgrobacterium rhizogenes as a vector for expressing genes in plant cellshas been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,1987.

[0059] Drug selection is generally used to select for cultured mammaliancells into which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems mayalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate.

[0060] Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell.

[0061] The recombinant islet cell antigen polypeptides expressed usingthe methods described herein are isolated and purified by conventionalprocedures, including separating the cells from the medium bycentrifugation or filtration, precipitating the proteinaceous componentsof the supernatant or filtrate by means of a salt, e.g. ammoniumsulfate, purification by a variety of chromatographic procedures, e.g.ion exchange chromatography or affinity chromatography, or the like.Methods of protein purification are known in the art (see generally,Scopes, R., Protein Purification, Springer-Verlag, NY (1982), which isincorporated herein by reference) and may be applied to the purificationof the recombinant proteins of the present invention. Substantially purerecombinant islet cell antigen polypeptides of at least about 50% ispreferred, at least about 70-80% more preferred, and 95-99% or morehomogeneity most preferred, particularly for pharmaceutical uses. Oncepurified, partially or to homogeneity, as desired, the recombinant isletcell antigen polypeptides may then be used diagnostically,therapeutically, etc. as further described below.

[0062] Recombinant 1851 polypeptides can also be produced by expressingislet cell antigen DNA fragments, such as fragments generated bydigesting an islet cell antigen cDNA at convenient restriction sites.The isolated recombinant polypeptides or cell-conditioned media are thenassayed for activity as described in the examples below. Alternatively,the proteins of the present invention may be synthesized followingconventional synthesis methods such as the solid-phase synthesis usingthe method of Barany and Merrifield (in The Peptides. Analysis,Synthesis, Biology Vol. 2, Gross and Meienhofer, eds, Academic Press,NY, pp. 1-284, 1980, which are incorporated herein by reference), bypartial solid-phase techniques, by fragment condensation or by classicalsolution addition. Short polypeptide sequences, or libraries ofoverlapping peptides, usually from about 6 up to about 35 amino acids,which correspond to selected islet cell antigen polypeptide regions canbe readily synthesized and then screened in screening assays designed toidentify peptides having a desired activity, such as domains which areresponsible for or contribute to binding activity, immunodominantepitopes (particularly those recognized by autoantibodies), and thelike.

[0063] Although the use of recombinant 1851 polypeptides is preferredwithin the methods of the present invention, 1851 polypeptides may alsobe prepared from cells that naturally produce 1851 protein (such asislet cells). For example, 1851 polypeptides may be prepared from isletcells by isolation of a membrane fraction. This 1851-enriched fractionis then used to detect autoantibodies to 1851 in prediabetic anddiabetic sera.

[0064] Islet cell antigen polypeptides produced according to the presentinvention can be used diagnostically, in the detection and quantitationof autoantibodies in a biological sample, that is, any sample derivedfrom or containing cells, cell components or cell products, including,but not limited to, cell culture supernatants, cell lysates, clearedcell lysates, cell extracts, tissue extracts, blood plasma, serum, andfractions thereof. By means of having islet cell antigen polypeptideswhich specifically bind to autoantibodies in prediabetic and diabeticsera, the presence or absence of such autoantibodies can be determined,and the concentration of such autoantibodies in an individual can bemeasured. This information can then be used to monitor the progressionor regression of the potentially harmful autoantibodies in individualsat risk of, or with a predisposition to develop IDDM, and would beuseful for predicting the clinical course of the disease in a patient.The assay results can also find use in monitoring the effectiveness oftherapeutic measures for treatment of IDDM or related diseases.

[0065] As will be recognized by those skilled in the art, numerous typesof immunoassays are available for use in determining the presence ofautoantibodies. For instance, direct and indirect binding assays,competitive assays, sandwich assays, and the like, as are generallydescribed in, e.g., U.S. Pat. Nos. 4,642,285; 4,376,110; 4,016,043;3,879,262; 3,852,157; 3,850,752; 3,839,153; 3,791,932; and Harlow andLane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,N.Y., 1988, each incorporated herein by reference. In one assay format,autoantibodies directed to the polypeptides of the present invention arequantified directly by measuring the binding of autoantibodies in abiological sample to recombinant or synthetic islet cell antigenpolypeptides. The biological sample is contacted with at least one isletcell antigen polypeptide of the invention under conditions conducive toimmune complex formation. The immune complexes formed between the isletcell antigen polypeptide and the antibodies are then detected, and thepresence and quantity of autoantibodies can then be used to diagnose ordirect treatment of IDDM. The immune complexes can be detected by meansof antibodies that bind to the islet cell antigen of the presentinvention or by labeling the polypeptide as described below. Separationsteps (e.g., washes) may be necessary in some cases to distinguishspecific binding over background. In another format, the serum level ofa patient's autoantibodies to the islet cell antigen polypeptides inserum can be measured by competitive binding with labeled or unlabeledantibodies to the islet cell antigen polypeptides of the presentinvention. Unlabeled 1851 polypeptides can be used in combination withlabeled antibodies that bind to human antibodies or to islet cellantigens. Alternatively, the islet cell antigen polypeptide can bedirectly labeled. A wide variety of labels can be employed, such asradionuclides, particles (e.g., gold, ferritin, magnetic particles, redblood cells), fluors, enzymes, enzyme substrates, enzyme cofactors,enzyme inhibitors, ligands (particularly haptens), chemiluminescers,biotin and other compounds that provide for the detection of the labeledpolypeptide or protein. For example, an 1851 polypeptide can beradiolabeled using conventional methods such as in vitro transcriptionand translation. Radiolabeled 1851 polypeptide is combined with patientserum under conditions suitable for immune complex formation. Immunecomplexes are then separated, such as by binding to protein A.Precipitated 1851 polypeptides are then quantitated by conventionalmethods, such as gel electrophoresis, fluorography, densitometry or bydirect counting of immunoprecipitated, radiolabeled antigen. The amountof 1851 polypeptide precipitated by test sera can be statisticallycompared to mean counts precipitated by healthy control sera, eachmeasured separately. In an alternative format, an 1851 polypeptideantigen, labeled with biotin, is combined with patient serum underconditions suitable for immune complex formation. The serum is thentransferred to a protein A-coated container, such as a well of an assayplate, and the container is allowed to stand so that immune complexescan form. The container is then washed, and streptavidin, conjugated toa suitable enzyme (e.g. alkaline phosphatase), is added. A chromogenicsubstrate is then added, and the presence of 1851 polypeptideautoantibodies in the sample is indicated by a color change. Additionalassay formats will be evident to those skilled in the art.

[0066] Thus, autoantibodies to islet cell antigen polypeptides can beidentified and, if desired, extracted from a patient's serum by bindingto 1851 polypeptides of the present invention. The islet cell antigenpolypeptides may be attached, e.g., by adsorption, to an insoluble orsolid support, such as ELISA microtiter well, microbead, filtermembrane, insoluble or precipitable soluble polymer, etc. to function asan affinity resin. The captured autoantibodies can then be identified byseveral methods. For example, antisera or monoclonal antibodies to theantibodies can be used. These antisera or monoclonal antibodies aretypically non-human in origin, such as rabbit, goat, mouse, etc. Theseanti-antibodies can be detected directly if attached to a label such as¹²⁵I, enzyme, biotin, etc., or can be detected indirectly by a labeledsecondary antibody made to specifically detect the anti-antibody.

[0067] The diagnostic methods of the present invention can be used inconjunction with other known assays and diagnostic techniques (see forexample, WO 95/07464, incorporated herein by reference in its entirety).Such other assays and techniques include measurement of body mass index(BMI), defined as the quotient of the patient's weight in kg divided bythe square of height in meters; C-peptide level (Heding, Diabetologia11: 541-548 (1975); Landin-Olsson et al., Diabetologia 33: 561-568(1990)); or one or more additional diabetes-associated autoantibodies,genotypes or loci. A low BMI (i.e. less than about 25) in combinationwith other indicators is suggestive of type I diabetes. BMI is thus auseful indicator for distinguishing type I from type II diabetes.C-peptide level can be measured using standard methods, such as that ofHeding (ibid.), in which insulin and proinsulin are removed from serumand C-peptide is measured in the resulting insulin-free fractionradioimmunologically.

[0068] The islet cell antigen polypeptides of the current invention canalso be used to assess T cell reactivity, as a method for monitoring thedisease state in a patient. Mammalian islet cell antigen 1851 peptideswill generally comprise at least about 12 amino acids, and more oftenfrom about 15 amino acids to about 20 or more amino acids. In someinstances, a substantial portion or domain or even the entire 1851protein, can be used to assess T cell reactivity in peripheral bloodmononuclear cells (PBMNCs) from prediabetics. Methods for detecting suchin vitro activity are known in the art, including a proliferation assaymeasuring ³H-thymidine incorporation, analysis of activation markers,such as CD69, or measuring cytokine production, such as IL-2.Correlations can be drawn between T cell reactivity to islet cellantigen 1851 and conversion from prediabetes to diabetes. Thiscorrelation would be consistent with the appearance of autoantibodies toislet cell antigen peptides late in prediabetes (Christie et al.,Diabetes 43:1254-59, 1994).

[0069] Mammalian cells, such as COS cells or L cells, may also betransfected with appropriate Class I or Class II alleles specific forthe islet cell antigen of the present invention. Such MHC molecules maybe soluble or membrane bound, and the 1851 antigenic polypeptide may berecombinantly tethered to the N-terminal region of the α or β chainusing a flexible linker containing, for example, repeating glycineresidues separated by a serine residue, such that the antigenic peptidebinds to the MHC molecule and is properly presented to the T cell.Alternatively, the antigenic peptide may be exogenously loaded into theMHC peptide binding grove. The MHC-antigenic peptide complex can then beused to assess the reactivity of peripheral blood T cells derived fromprediabetic or diabetic patients. This reactivity may be assessed bymethods known in the art, such as ³H thymidine incorporation, cytokineproduction or cytolysis. Alternatively, islet cell antigen expressed inmicroorganisms can be “fed” to peripheral blood mononuclear cells(PBMN). The antigen-fed cells can then be used to stimulate peripheralblood T cells derived from diabetics or prediabetics.

[0070] The islet cell antigen polypeptides are also contemplated to beadvantageous for use as immunotherapeutics to induce immunologicaltolerance or nonresponsiveness (anergy) to 1851 polypeptide autoantigensin patients predisposed or already mounting an immune response to 1851polypeptide autoantigens of the islet β-cells. This therapy can take theform of autoantigenic 1851 peptides bound to an appropriate MHC Class Ior Class II molecule as described above. The therapy can also be in theform of oral tolerance (Weiner et al., Nature 376: 177-80, 1995), or IVtolerance, for example. The use of polypeptide antigens in suppressionof autoimmune disease is disclosed by Wraith, et al., (Cell 59: 247-55,1989). Tolerance can be induced in patients, although conditions forinducing such tolerance will vary according to a variety of factors. Ina neonate, tolerance can be induced by parenteral injection of an isletcell antigenic polypeptide, either with recombinant polypeptide orsynthetic antigen, or more conveniently by oral administration in anappropriate formulation. The precise amount of administration, its modeand frequency of dosages will vary.

[0071] To induce immunological tolerance to the islet cell autoantigensin an adult susceptible to or already suffering from a islet cellantigen related disease such as IDDM, the precise amounts and frequencyof administration will also vary, for adults about 1 to 1,000 mg/kg canbe administered by a variety of routes, such as parenterally, orally, byaerosols, intradermal injection, etc. For neonates the doses willgenerally be higher than those administered to adults; e.g. 100 to 1,000mg/kg.

[0072] The islet cell antigen 1851 polypeptides will typically be moretolerogenic when administered in a soluble form rather than anaggregrated or particulate form. Persistence of an islet cell antigenpolypeptide of the invention is generally needed to maintain tolerancein an adult, and thus may require more frequent administration of theantigen, or its administration in a form which extends the half-life ofthe islet cell antigen. See for example, Sun et al. (Proc. Natl. Acad.Sci. USA 91: 10795-99, 1994).

[0073] The islet cell antigen polypeptides described herein are alsocontemplated to be advantageous for use as immunotherapeutics intreating longer term IDDM patients that have been identified byautoantibody testing at the time of clinical non-insulin dependentdiabetes mellitus (NIDDM) diagnosis. Intervention in these patients maybe especially effective, perhaps due to the slowly progressive nature oftheir β cell destruction. Since the numbers of such patients is nearlythe same as those with classical childhood IDDM, there is a need forsuch therapeutic intervention (Hagopian et al., J. Clin. Invest.91:368-74; 1993; Harris and Robbins, Diabetes Care 17:1337-40, 1994; andKobayashi et al., Diabetes 45:622-26, 1996).

[0074] The N-terminal domain of islet cell antigen 1851 is expected tobe inside the insulin secretory granule. The islet cell antigenpolypeptides of the current invention contain post translationalmodification sites within the N-terminal domain. A dibasic site ortribasic site at amino acid residues 228-230 (Arg-Lys-Lys) in SEQ IDNO:22 and amino acid residues 422-424 (Arg-Lys-Lys) in SEQ ID NO:16could result in cleavage of a 420 amino acid post-translationallymodified mammalian islet cell antigen polypeptide from the islet cellantigen 1851 polypeptide. All or part of this cleaved polypeptide may bereleased from the β cell via either the constitutive secretory pathwayfor granule halo components, or via the regulated pathway involved ininsulin release. Detection and quantitation of post translationallymodified polypeptides in a biological sample (that is, any samplederived from or containing cells, cell components or cell products,including, but not limited to, cell culture supernatants, cell lysates,cleared cell lysates, cell extracts, tissue extracts, blood plasma,serum, and fractions thereof) can be used diagnostically to monitordisease state in a patient. The presence or absence of such polypeptidesin prediabetic and diabetic sera can be determined, for example byradioimmunoassay, and the concentration of such polypeptides in such anindividual serum sample can be measured. This information can then beused, for example, to monitor insulin secretory activity, such as β cellinsulin secretory rates; or to indicate altered β cell physiologyassociated with cellular stress as in an immune attack. Peptide levelscould be an indicator of β cell distress or β cell death, and would beuseful for predicting the disease state in a patient. Alternatively, thepeptides herein function serve in paracrine or endocrine signaling toother islet cells or remote cells in other organs. The assay results canalso find use in monitoring the effectiveness of therapeutic measuresfor treatment of IDDM or related diseases. In a preferred embodiment, apost-translationally modified mammalian islet cell antigen polypeptidecomprises the sequence of SEQ ID NO:22 from His, amino acid residue 1 toGlu, amino acid residue 227. In another preferred embodiment thebiological sample is blood.

[0075] The present invention also relates to a pharmaceuticalcomposition comprising an islet cell antigen polypeptide of the presentinvention, together with a pharmaceutically acceptable carrier orvehicle, such as saline, buffered saline, water or the like.Formulations may further include one or more excipients, preservatives,solubilizers, etc. Methods of formulation are well known in the art andare disclosed, for example, in Remington's Pharmaceutical Sciences,Gennaro, ed., Mack Publishing Co., Easton Pa., 1990, which isincorporated herein by reference. Therapeutic doses will generally be inthe range of 0.1 to 100 μg/kg of patient weight, with the exact dosedetermined by the clinician according to accepted standards, taking intoaccount the nature and severity of the condition to be treated, patienttraits, etc. Determination of dose is within the level of ordinary skillin the art. In general, a therapeutically effective amount of an isletcell antigen polypeptide of the present invention is an amountsufficient to produce a clinically significant reduction in β-cell lossor a delay of clinical onset of IDDM.

[0076] In a related aspect, the present invention provides diagnostickits for use with the recombinant or synthetic islet cell antigenpolypeptides of the present invention, in detecting autoantibodies topancreatic β-islet cells. Thus, 1851 polypeptides may be provided,usually in lyophilized form, in a container, either alone or inconjunction with additional reagents, such as 1851-specific antibodies,labels, and/or anti-human antibodies and the like. The 1851 polypeptidesand antibodies, which may be conjugated to a label or unconjugated, areincluded in the kits with buffers, such as Tris phosphate, carbonate,etc., stabilizers, biocides, inert proteins, e.g., serum albumin, andthe like. Frequently it will be desirable to include an inert extenderor excipient to dilute the active ingredients, where the excipient maybe present in from about 1 to 99% of the total composition. Where anantibody capable of binding to the islet cell antigen polypeptideautoantibody or to the recombinant or synthetic 1851 polypeptide isemployed in an assay, this will typically be present in a separate vial.

[0077] Within one aspect of the present invention, islet cell antigenpolypeptides, including derivatives thereof, as well as portions orfragments of these polypeptides, are utilized to prepare antibodies fordiagnostic or therapeutic uses which specifically bind to islet cellantigen polypeptides. As used herein, the term “antibodies” includespolyclonal antibodies, monoclonal antibodies, antigen-binding fragmentsthereof such as F(ab′)₂ and Fab fragments, as well as recombinantlyproduced binding partners. These binding partners incorporate thevariable regions from a gene which encodes a specifically bindingmonoclonal antibody. Antibodies are defined to be specifically bindingif they bind to the islet cell antigen polypeptides with a K_(a) ofgreater than or equal to 10⁷/M. The affinity of a monoclonal antibody orbinding partner may be readily determined by one of ordinary skill inthe art (see, Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949).

[0078] Methods for preparing polyclonal and monoclonal antibodies havebeen well described in the literature (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal HybridomaAntibodies: Techniques and Applications, CRC Press, Inc., Boca Raton,Fla., 1982, which is incorporated herein by reference) As would beevident to one of ordinary skill in the art, polyclonal antibodies maybe generated from a variety of warm-blooded animals such as horses,cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, for example.The immunogenicity of the islet cell antigen polypeptide may beincreased through the use of an adjuvant such as Freund's complete orincomplete adjuvant. A variety of assays known to those skilled in theart may be utilized to detect antibodies which specifically bind to anislet cell antigen. Exemplary assays are described in detail inAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1988. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immuno-sorbent assays, dotblot assays, inhibition or competition assays, and sandwich assays.

[0079] Additional techniques for the preparation of monoclonalantibodies may be utilized to construct and express recombinantmonoclonal antibodies. Briefly, mRNA is isolated from a B cellpopulation and used to create heavy and light chain immunoglobulin cDNAexpression libraries in a suitable vector such as the λIMMUNOZAP(H) andλIMMUNOZAP(L) vectors, which may be obtained from Stratogene CloningSystems (La Jolla, Calif.). These vectors are then screened individuallyor are co-expressed to form Fab fragments or antibodies (Huse et al.,Science 246: 1275-81, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA86: 5728-32, 1989). Positive plaques are subsequently converted to anon-lytic plasmid which allows high level expression of monoclonalantibody fragments in E. coli.

[0080] Binding partners such as those described above may also beconstructed utilizing recombinant DNA techniques to incorporate thevariable regions of a gene which encodes a specifically bindingantibody. The construction of these proteins may be readily accomplishedby one of ordinary skill in the art (see for example, Larrick et al.,Biotechnology 7: 934-38, 1989; Reichmann et al., Nature 322: 323-27,1988 and Roberts et al. Nature 328: 731-34, 1987). Once suitableantibodies or binding partners have been obtained, they may be isolatedor purified by many techniques well described in the literature (see forexample, Antibodies: A Laboratory Manual, ibid.). Suitable techniquesinclude protein or peptide affinity columns, HPLC or RP-HPLC,purification on protein A or protein G columns or any combination ofthese techniques. Within the context of the present invention, the term“isolated” as used to define antibodies or binding partners means“substantially free of other blood components.”

[0081] Antibodies of the present invention may be produced by immunizingan animal, a wide variety of warm-blooded animals such as horses, cows,goats, sheep, dogs, chickens, rabbits, mice, and rats can be used, witha recombinant or synthetic islet cell antigen polypeptide or a selectedportion thereof (e.g., a peptide). For example, by selected screeningone can identify a region of the islet cell antigen polypeptide such asthat predominantly responsible for recognition by anti-islet cellantigen polypeptide antibodies, or a portion which comprises an epitopeof a islet cell antigen polypeptide variable region, which may thusserve as a islet cell antigen polypeptide-specific marker. Antibodyproducing cells obtained from the immunized animals are immortalized andscreened, or screened first for, e.g., the production of antibody whichinhibits the interaction of the anti-islet cell antigen polypeptideautoantibody with the islet cell antigen polypeptide and thenimmortalized. As the generation of human monoclonal antibodies to ahuman antigen, such as an 1851 polypeptide, may be difficult withconventional immortalization techniques, it may be desirable to firstmake non-human antibodies and then transfer via recombinant DNAtechniques the antigen binding regions of the non-human antibodies, e.g.the F(ab′)2 or hypervariable regions, to human constant regions (Fc) orframework regions to produce substantially human molecules. Such methodsare generally known in the art and are described in, for example, U.S.Pat. No. 4,816,397, and EP publications 173,494 and 239,400, which areincorporated herein by reference.

[0082] Alternatively, one may isolate DNA sequences which encode a humanmonoclonal antibody or portions thereof that specifically bind to isletcell antigen polypeptides by screening a DNA library from human B cellsaccording to the general protocol outlined by Huse et al., Science 246:1275-81, 1989, incorporated herein by reference, and then cloning andamplifying the sequences which encode the antibody (or binding fragment)of the desired specificity.

[0083] In another aspect of the invention, the mammalian islet cellantigen polypeptides can be used to clone T cells which have specificreceptors for the islet cell antigen polypeptide. Once the islet cellantigen polypeptide specific T cells are isolated and cloned usingtechniques generally available to the skilled artisan, the T cells ormembrane preparations thereof can be used to immunize animals to produceantibodies to the islet cell antigen polypeptide receptors on T cells.The antibodies can be polyclonal or monoclonal. If polyclonal, theantibodies can be murine, lagomorph, equine, ovine, or from a variety ofother mammals. Monoclonal antibodies will typically be murine in origin,produced according to known techniques, or human, as described above, orcombinations thereof, as in chimeric or humanized antibodies. Theanti-islet cell antigen polypeptide receptor antibodies thus obtainedcan then be administered to patients to reduce or eliminate T cellsubpopulations which recognize and participate in the immunologicaldestruction of islet cell antigen polypeptide bearing cells in anindividual predisposed to or already suffering from a disease, such asIDDM. Further, the islet cell antigen polypeptide T cell receptors canthus be identified, cloned and sequenced, and receptor polypeptidessynthesized which bind to the islet cell antigen polypeptides and blockrecognition of the islet cell antigen polypeptide-bearing cells, therebyimpeding the autoimmune response against host islet cells. Howell et al.(Science 246: 668-70, 1989) have demonstrated that T cell receptorpeptides can block the formation of the tri-molecular complex between Tcells, autoantigen and major histocompatibilty complex in an autoimmunedisease model.

[0084] Antibodies and binding partners of the present invention may beused in a variety of ways. The tissue distribution of the islet cellantigen, for example, may be determined by incubating tissue slices witha labeled monoclonal antibody which specifically binds to the islet cellantigen polypeptides, followed by detection of the presence of the boundantibody. Labels suitable for use within the present invention are wellknown in the art and include, among others, fluorescein, isothiocyanate,phycoerythrin, horseradish peroxidase, and colloidal gold. Theantibodies of the present invention may also be used for thepurification of the islet cell antigen polypeptides of the presentinvention. The coupling of antibodies to solid supports and their use inpurification of proteins is well known in the literature (see forexample, Methods in Molecular Biology, Vol. 1, Walker (Ed.), HumanaPress, New Jersey, 1984, which is incorporated by reference herein inits entirety). Antibodies of the present invention may be used as amarker reagent to detect the presence of islet cell antigen polypeptideson cells or in solution. Such antibodies are also useful for westernanalysis or immunoblotting, particularly of purified cell secretedmaterial. Polyclonal, affinity purified polyclonal, monoclonal andsingle chain antibodies are suitable for use in this regard. Inaddition, proteolytic and recombinant fragments and epitope bindingdomains can be used herein. Chimeric, humanized, veneered, CDR-replaced,reshaped or other recombinant whole or partial antibodies are alsosuitable.

[0085] The following examples are offered by way of illustration, not byway of limitation.

EXAMPLES Example 1 Synthesis of Macaque Islet Cell cDNA and Preparationof a Macaque Islet Cell cDNA Library

[0086] Islets of Langerhans (˜100,000) were isolated by collagenasedigestion and Ficoll density gradient centrifugation from pancreas ofMacaca nemestrina (obtained from the University of Washington PrimateCenter, Seattle, Wash.). These cells were then flash frozen in liquidnitrogen and stored at −80° C. until use. Total RNA from the islets wasisolated according to the method of Chirgwin et al., Biochemistry 18:52-94, 1994, incorporated herein by reference, using polytronhomogenization in guanidinium thiocynate and LiCl centrifugation.Poly(A)+ RNA was isolated using oligo d(T) cellulose chromatography(Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-12, 1972).

[0087] First strand cDNA was synthesized from two-time polyd(T)-selected liver poly(A)⁺RNA. Ten microliters of a solutioncontaining 10 μg of liver poly(A)⁺RNA was mixed with 2 μl of 20 pmole/μlfirst strand primer ZC3747 (SEQ ID NO:8) and 4 μl ofdiethylpyrocarbonate-treated water. The mixture was heated at 65° C. for4 minutes and cooled by chilling on ice.

[0088] The first strand cDNA synthesis was initiated by the addition of8 μl of 5× SUPERSCRIPT buffer (GIBCO BRL, Gaithersburg, Md.), 4 μl of100 mM dithiothreitol, and 2.0 μl of a deoxynucleotide triphosphatatesolution containing 10 mM each of dATP, dGTP, dTTP and 5-methyl-dCTP(Pharmacia LKB Biotechnology Inc., Piscataway, N.J.) to the RNA-primermixture. The reaction mixture was incubated at 45° C. for 4 minutes.After incubation, 10.0 μl of 200 U/μl SUPERSCRIPT reverse transcriptase(GIBCO BRL) was added. The efficiency of the first strand synthesis wasanalyzed in a parallel reaction by the addition of 10 μCi of ³²P-αdCTPto a 5 μl aliquot of the reaction mixture to label the reactionproducts. The first strand synthesis reaction mixtures were incubated at45° C. for 45 minutes followed by a 15 minute incubation at 50° C.Unincorporated nucleotides were removed from each reaction byprecipitating the cDNA in the presence of 8 μg of glycogen carrier, 2.5M ammonium acetate and 2.5 volume ethanol. The unlabeled cDNA wasresuspended in 50 μl water and used for the second strand synthesis. Thelength of first strand cDNA was assessed by resuspending the labeledcDNA in 20 μl water and determining the cDNA size by agarose gelelectrophoresis.

[0089] Second strand synthesis was performed on the RNA-DNA hybrid fromthe first strand synthesis reaction under conditions that promoted firststrand priming of second strand synthesis resulting in DNA hairpinformation. A reaction mixture was prepared containing 20.0 μl of 5×polymerase I buffer (100 mM Tris, pH 7.4, 500 mM KCl, 25 mM MgCl_(2, 50)MM (NH₄)₂SO₄), 1.0 μl of 100 mM dithiothreitol, 2.0 μl of a solutioncontaining 10 mM of each deoxynucleotide triphosphate, 3.0 μl 5 mMα-NAD, 1.0 μl of 3 U/μl E. coli DNA ligase (New England Biolabs, Inc.,Beverly, Mass.), 5.0 μl of 10 U/μl E. coli DNA polymerase (Gibco BRL)and 50.0 μl of the unlabeled first strand DNA. A parallel reaction inwhich a 10 μl aliquot of the second strand synthesis was labeled by theaddition of 10 μCi of ³²P-αdCTP was used to monitor the efficiency ofsecond strand synthesis. The reaction mixtures were incubated at roomtemperature for 5 minutes followed by the addition of 1.5 μl of 2 U/μlRNase H (Gibco BRL) to each reaction mixture. The reactions wereincubated at 15° C. for 2 hours and 15 minutes, followed by a 15 minuteincubation at room temperature. The reactions were each terminated byserial phenol/chloroform and chloroform/isoamylalcohol extractions. TheDNA from each reaction was precipitated in the presence of ethanol and2.5 M ammonium acetate. The DNA from the unlabeled reaction wasresuspended in 100 μl water. The labeled DNA was resuspended andelectrophoresed as described above.

[0090] The single-stranded DNA in the hairpin structure was cleavedusing mung bean nuclease. The reaction mixture contained 20 μl of 10×Mung Bean Nuclease Buffer (Stratagene Cloning Systems, La Jolla,Calif.), 16 μl of 100 mM dithiothreitol, 54 μl water, 100 μl of thesecond strand cDNA, and 10 μl of a 1:10 dilution of Mung Bean Nuclease,final concentration 10.5 U/μl (Promega Corp., Madison, Wis.) inStratagene MB dilution Buffer (Stratagene Cloning Systems). The reactionwas incubated at 37° C. for 15 minutes, and the reaction was terminatedby the addition of 20 μl of Tris-HCl, pH 8.0 followed by sequentialextractions with phenol/chloroform and chloroform/isoamylalcohol.Following the extractions, the DNA was precipitated in ethanol andresuspended in water.

[0091] The resuspended cDNA was blunt-ended with T4 DNA polymerase. ThecDNA, which was resuspended in a volume of 50 μl of water, was mixedwith 50 μl of 5× T4 DNA polymerase buffer. (250 mM Tris-HCl, pH 8.0, 250mM KCl, 25 MM MgCl₂), 3 μl of 100 mM dithiothreitol, 3 μl of a solutioncontaining 10 mM of each deoxynucleotide triphosphate, and 4 μl of 1.0U/μl T4 DNA polymerase (Boehringer Mannheim, Indianapolis, Ind.). Afteran incubation at 10° C. for 60 minutes, the reaction was terminated byserial phenol/chloroform and chloroform/isoamylalcohol extractions. ThecDNA fragments less than 400 bp in length were removed by chromatographyon a Clontech TE400 spin column (Clontech, Palo Alto, Calif.). The DNAwas ethanol precipitated and resuspended in 9 μl of water. Based on theincorporation of ³²P-dCTP, the yield of cDNA was estimated to be 4 μgfrom a starting mRNA template of 10 μg.

[0092] Eco RI adapters (Pharmacia LKB Biotechnology Inc., Piscataway,N.J.) were added to the cDNA prepared above to facilitate the cloning ofthe cDNA into a mammalian expression vector. A 9 μl aliquot of the cDNAand 975 pmole of the adapter (15 μl) were mixed with 3 μl 10× ligasebuffer (Promega Corp.), 1 μl 10 mM ATP, and 20 Units (2 μl), of T4 DNAligase. (Promega Corp.). The reaction was incubated for 16 hours at atemperature gradient of 4° C. to 15° C. The reaction was terminated bythe addition of 185 μl water, 25 μl REACT 2 buffer (Gibco BRL) followedby an incubation at 65° C. for between 30 and 60 minutes. Afterincubation, the reaction was terminated by serial phenol/chloroform andchloroform/isoamylalcohol extractions and ethanol precipitation asdescribed above. Following centrifugation, the DNA pellet was washedwith 70% ethanol and was air dried. The pellet was resuspended in 89 μlof water.

[0093] To facilitate the directional insertion of the cDNA into amammalian expression vector, the cDNA was digested with Xho I, resultingin a cDNA having a 5′ Eco RI adhesive end and a 3′ Xho I adhesive end.The Xho I restriction site at the 3′ end of the cDNA was introducedthrough the ZC3747 primer (SEQ ID NO:8). The restriction digestion wasterminated by serial phenol/chloroform and chloroform/isoamylalcoholextractions. The cDNA was ethanol precipitated, and the resulting pelletwas washed with 70% ethanol and air-dried. The pellet was resuspended in1× loading buffer (10 mM phosphate buffer, pH 8.8, 5% glycerol, 0.125%bromphenol blue).

[0094] The resuspended cDNA was heated to 65° C. for 10 minutes, cooledon ice and electrophoresed on a 0.8% low melt agarose gel (Seaplaque GTGLow Melt Agarose, FMC Corp., Rockland, Me.) using a 1 Kb ladder (GibcoBRL) as size markers. The contaminating adapters and by-productfragments below 600 bp in size were excised from the gel. The electrodeswere reversed, and the cDNA was electrophoresed until concentrated nearthe lane origin. The area of the gel containing the concentrated DNA wasexcised, placed in a microfuge tube, and the approximate volume of thegel slice was determined. An aliquot of TE (10 mM Tris HCl pH 7.4, 1 mMdisodium ethylenediaminetetraacetate.2 H₂O (EDTA)) equivalent to halfthe volume of the gel slice was added to the tube, and the agarose wasmelted by heating to 65° C. for fifteen minutes. Following equilibrationof the sample to 42° C., approximately 5 units of β-Agarase I (NewEngland Biolabs, Inc.) was added. The sample was incubated for 2 hoursto digest the agarose. After incubation, a 0.1× volume of 3M sodiumacetate was added to the sample, and the mixture was incubated on icefor fifteen minutes. After incubation, the sample was centrifuged at14,000×g for 10 minutes to remove the undigested agarose. The cDNA inthe supernatant was ethanol precipitated. The cDNA pellet was washedwith 70% ethanol, air dried and resuspended in 37 μl of water. The cDNArecovered from the agarose gel was phosphorylated using T4polynucleotide kinase. The reaction consisted of 37 μl cDNA, 5 μl 10×Stratagene Ligase Buffer (Stratagene Cloning Systems). Following a 5minute incubation at 65° C., the reaction was cooled to room temperaturewhere 5 μl 10 mM ATP (Pharmacia) and 3 μl T4 DNA polymerase (10 U/μl,Stratagene) were added. The reaction was incubated at 37° C. for 45minutes and at 65° C. for 10 minutes. The reaction was terminated byserial phenol/chloroform extractions. The samples were chromatographedthrough a Clontech TE400 spin column and were precipitated in thepresence of 2.5 M ammonium acetate. The cDNA was resuspended in 15 μl of2.5 mM Tris-HCl, pH 8.0, 0.25 mM EDTA.

[0095] The resulting Eco RI-Xho I cDNA library was cloned into the E.coli vector pZCEP (Jelinek et al., Science 259: 1614-16, 1993). EcoRI-Xho I linearized pZCEP was ligated with the Eco RI-Xho I cDNAlibrary. The resulting plasmids were electroporated into the E. colistrain DH10B ELECTROMAX˜ (Gibco BRL). The library was plated to obtain>5×10⁵ independent colonies and aliquoted into 120 pools to giveapproximately 5,000 colonies per pool. An aliquot of the cells from eachpool was removed for use in preparing plasmid DNA. The remaining cellmixtures were brought to a final concentration of 15% glycerol,aliquoted and frozen at −80° C. Plasmid DNA was prepared from each pooland the resulting plasmid DNA was digested with RNAse (BoehringerMannheim) according to the manufacturer's instructions. The RNAsereaction was terminated by a phenol/chloroform/isoamylalcohol (24:24:1)extraction, and the DNA was ethanol precipitated. The pools weresystematically screened as described in the examples below.

Example 2 Transfection of Macaque DNA into COS-7 Cells

[0096] Macaque DNA from each pool was transfected into COS-7 cells(African Green Monkey Kidney cells, ATCC CRL 1651) using the methodessentially described by McMahan et al. (EMBO J. 10: 2821-32, 1991;which is incorporated by reference herein in its entirety). Briefly, oneday prior to transfection approximately 2×10⁵ COS-7 cells in 2 ml growthmedium containing 10% fetal bovine serum (Dulbecco's modified Eagle'smedium (DMEM), 1% L-glutamine, 1% PNS antibiotic mix (Gibco BRL), 25 mMHepes, and 1 mM NaPyruvate) were plated on sterile, single-chamberslides (Nunc AS, Roskilde, Denmark) that had been coated with 10 μg/mlof human fibronectin in PBS for 30 minutes at 37° C. and washed withphosphate buffered saline (PBS). For each pool to be tested, 1-2 μg ofmacaque islet cell library pooled DNA was added into 100 μl of serumfree medium (SFM, F/DV medium, 10 mg/l transferrin, 2 μg/l selenium, 10mg/l fetuin, 5 mg/l insulin, 1 L-glutamine, 25 mM Hepes, 1 mMNaPyruvate, and 0.1 mM NEAA). To each DNA sample was added 100 μl SFMcontaining 12 μl LipofectAMINE˜ (Gibco BRL). The transfection solutionwas mixed by pipetting up and down and kept at room temperature for 15to 45 minutes. To each mix was added 0.8 ml SFM which was then gentlyadded to the COS-7 cells which had been washed once with SFM. The cellswere incubated at 37° C., 5% CO₂ for 4-5 hours. One milliliter of growthmedium containing 20% FBS was added to each slide. Slides were incubatedovernight at 37° C., 5% CO₂. The spent medium was removed and replacedwith 2 ml growth medium containing 10% FBS and the cells incubated for24 to 48 hours, preferably 48 hours, at 37° C., 5% CO₂.

Example 3 Diabetic Sera

[0097] Sera from two prediabetic subjects, EmWi and JoGr, were selectedfor screening the islet cell cDNA library. Sera from both subjects werecharacterized for autoantibodies to known β-cell antigens usingtechniques known in the art. The sera were tested for GAD65autoantibodies using an in vitro transcription/translation assay (Grubinet al., Diabetologia 37: 344-50, 1994) followed by immunoprecipitationusing radiolabeled recombinant human GAD65 according to Hagopian et al.,J. Clin. Invest. 91: 368-74, 1993.

[0098] Recombinant radiolabeled GAD was expressed in the presence of ³⁵SMethionine (Amersham Corp., Arlington Heights, Ill.) using the Sp6bacteriophage promoter and the TNT reticulocyte lysate kit (Promega),according to manufacturer's direction. ³⁵S Methionine incorporation wasdetermined by precipitation using trichloroacetic acid (TCA), and 25% ormore incorporation was considered acceptable. Radiolabeled antigen wasstored at −80° C. until use.

[0099] Radiolabeled antigen was diluted 1:10 in immunoprecipitationbuffer (150 mM NaCl, 1% v/v Triton X-114 (Sigma Chemical Co., St. Louis,Mo.), 0.05% Bovine serum albumin (Sigma), 10 mM benzamidine (Sigma), and10 mM HEPES pH 7.4). The antigen was incubated for preclearing for 4hours at 4° C. with 50 μl normal human serum. Immunoglobulin was removedusing 200 μl Protein A Sepharose beads (Pharmacia LKB BiotechnologyInc.) for 45 minutes. The cleared supernatant was diluted to 50,000TCA-precipitable counts per minute (cpm) per 400 μl immunoprecipitationbuffer. Four microliters of serum from diabetic or control patients wasseparately incubated in duplicate with 400 μl diluted antigen at 4° C.overnight with mixing by gentle rotation. Antigen-antibody complexeswere precipitated by 16 μl Protein A Sepharose, and the pellet waswashed 5 times in ice-cold wash buffer which consisted of 10 mM HEPES pH7.4, 150 mM NaCl, 0.05% BSA, and 0.25% Triton X-114. Antigen wasdissociated from the pellet by boiling in the presence of 2% SDS and 5%β-mercaptoethanol, and counted by scintillation counting inscintillation fluid. Counts per minute reflect the level ofautoantibodies present in the sera to capture the antigen.

[0100] Autoantibodies to the protein tyrosine phosphatase IA-2/ICA512were detected as above using a radiolabeled cytoplasmic domain of humanIA-2/ICA512 (Lan et al., DNA Cell Biology 13: 505-14, 1994; and Hagopianet al., Autoimmunity 21: 61, 1995). The complete cytoplasmic domain ofhuman IA-2 was isolated by RT-PCR from U87MG glioblastoma cells (ATCCM85). Briefly, total RNA was prepared from 5×10⁷ glioblastoma cellswhich were homogenized in 3.5 ml guanidine/LiCl followed by CsClcentrifugation. First strand cDNA was synthesized using a Superscript˜Preamplification System (GIBCO BRL) according to the manufacturer'sdirections. One and one half microliters of a solution containing 5 μgtotal U87MG RNA was mixed with 1 μl oligo dT solution and 11.5 μldiethylpyrocarbonate-treated water. The mixture was heated at 70° C. for10 minutes and cooled by chilling on ice.

[0101] First strand cDNA synthesis was initiated by the addition of 2 μlSuperscript˜ II buffer, 2 μl 0.1 M dithiothreitol, 1 μl deoxynucleotidetriphosphate solution containing 10 mM each of dATP, dGTP, dTTP, anddCTP, and 1 μl of 200 U/μl Superscript˜ II reverse transcriptase to theRNA-primer mixture. The reaction was incubated at room temperature for10 minutes followed by an incubation at 42° C. for 50 minutes, then 70°C. for 15 minutes, then cooled on ice. The reaction was terminated byaddition of 1 μl RNase H which was incubated at 37° C. for 20 minutes,then cooled on ice.

[0102] A 100 μl PCR reaction mixture was then prepared containing 20 μlof first strand template, 8 μl 10× synthesis buffer, 3.3 μM ZC8802 (SEQID NO:9, contains 5′ Xho I site and ATG), 5.4 μM ZC8803 (SEQ ID NO:10,contains Eco RI site following stop codon), 65 μl dH₂O and 1 wax bead(AmpliWax˜, Perkin-Elmer Cetus, Norwalk, Conn.). Following an initialcycle of 95° C. for 2 minutes, 4° C. for 10 minutes, 5 U Taq polymerasewas added, and the reaction was amplified for 30 cycles of 1 minute at95° C., 2 minutes at 55° C. and 3 minutes at 72° C. The reaction mixturewas then stored at 4° C. The resulting 1.2 kb fragment (SEQ. ID. No.30)was digested with Eco RI-Xho I, treated with RNAse, then isolated by lowmelt agarose gel electrophoresis and ligated into Eco RI-Xho Ilinearized pZCEP. Sera were screened for IA-2/ICA512 autoantibodies asdescribed above for GAD autoantibodies.

[0103] Both EmWi and JoGr sera showed reactivity to IA-2/ICA512. Thesera were titered for IA-2/ICA512 reactivity on vector only transfectedCOS-7 cells using techniques known in the art, see for example,Greenbaum et al. (Diabetes 41: 1570-1574, 1992). The sera wereseparately adsorbed with porcine insulin (Hoechst, 10 mg/ml) and GAD (1mg/ml) until reactivity was abolished in the respective antibody assays.These sera were then retitered for IA-2/ICA512 as above. JoGr hadIA-2/ICA512 reactivity of 280 JDFU (Juvenile Diabetes Foundation Units)which persisted at >130 JDFU after adsorption. EmWi had IA-2/ICA512reactivity of 140 JDFU which persisted at >130 JDFU after adsorption.EmWi had the lowest background staining and was therefore used forprimary screening.

[0104] Twenty milliliters of EmWi was diluted 1:1 in 0.1 M NaPO₄ buffer,pH 8.0 and incubated with an equal volume of Protein A covalently linkedto Sepharose beads (Zymed, South San Francisco, Calif.) for affinitypurification. After gentle mixing for 45 minutes at 4° C., the slurrywas loaded onto a column and washed with 10 column volumes of 0.1 MNaPO₄ buffer, pH 8.0 and one column volume of 0.01 M NaPO₄ buffer, pH8.0, before elution of immunoglobulins with 0.05 M Na citrate buffer, pH3.5. Eluted immunoglobulins were immediately neutralized to pH 7.0 with2 M Tris, pH 8.0. Eluted fractions were evaluated by spectrophotometricabsorption at 280 nM, and peak fractions were pooled, aliquoted andflash frozen for storage at −80° C. Typically the concentration was 4mg/ml IgG. COS-7 cells were grown to confluence in 150 ml T-flasks,washed with PBS, fixed in 4% paraformaldehyde, and permeabilized byfreeze/thaw. The pooled sera were diluted to 1 mg/ml in PBS andincubated with the permeabilized COS-7 cell lysate overnight at 4° C.Supernatant was cleared at 100,000×g and aliquotted for storage at −80°C. for use in the binding assay.

Example 4 Binding Assay

[0105] The macaque DNA transformed COS-7 cells on single chamber slides,from Example 2, were prepared for assay by removing spent medium fromthe slides and washing the cells 3 times in PBS at room temperature. Thecells were fixed with 1 ml 50% ETOH/50% acetone for 5 minutes at roomtemperature followed by two washes in PBS and two washes in 1% bovineserum albumin (BSA) in PBS. The precleared serum (EmWi) was diluted to0.2 mg/ml in a 5% BSA in PBS solution, and 500 μl was added to each ofthe slides which were then covered, wrapped in plastic wrap, and rockedgently on a rocker overnight at room temperature.

[0106] The slides were then washed three times in a 1% BSA/PBS solution,three minutes for each wash. Following the final wash, the slides wereblocked for 10 minutes with 1 ml 5% BSA/4% normal goat serum (Sigma) inPBS at room temperature. The blocking buffer was removed, and 500 μl of0.02 mg/ml biotinylated Protein A (Amersham Corp., Arlington Heights,Ill.) in 5% BSA/4% normal goat serum/PBS was added, followed by a 30minute incubation at room temperature. The slides were washed threetimes with 1% BSA/PBS, three minutes for each wash, then 500 μlstreptavidin-gold (Amersham) diluted 1:50 in 5% BSA/4% normal goatserum/PBS was added to each slide. Following a 60 minute incubation atroom temperature the slides were washed three times in 1% BSA/PBS andone final time in PBS. The slides were then fixed by adding 0.5 ml of 9%formaldehyde/45% acetone in PBS for 30 seconds followed by three, 3minute washes in dH₂O.

[0107] An equal volume of silver enhancement solution and initiator(IntenSE˜ M Silver Enhancement Kit, Amersham) were mixed in a 15 mlconical tube, and 0.5 ml was added to each slide. The slides wereallowed to develop for 20 minutes or until the desired color intensitywas achieved. The slides were then rinsed twice for five minutes in dH₂Oand air dried. A single positive pool (#18) containing approximately5,000 clones was found out of approximately 50 pools screened using EmWisera.

[0108] To isolate the positive clone(s) from pool #18, one 150 mm platewas plated to give approximately 10,000 colonies from the #18 pool.Filter lifts were prepared using the methods essentially described byHanahan and Meselson (Gene 10: 63, 1980) and Maniatis et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982), which areincorporated herein by reference in their entirety. The hybridizationprobe was obtained by PCR amplification of plasmid DNA from pool #18.Briefly, an aliquot of the plasmid DNA from pool #18 was subjected toPCR amplification using oligonucleotides ZC8802 and ZC8803 (SEQ ID NOS:9and 10, respectively). A 50 μl reaction mixture was prepared containing0.05 μg of the plasmid DNA from pool #18; 20 pmole of ZC 8802 and ZC8803 (SEQ ID NOS:9 and 10, respectively); 10 nmoles of eachdeoxynucleotide triphosphate (Pharmacia); 4 μl 10× synthesis buffer(Boehringer Mannheim), and 2.5 U Taq polymerase (Boehringer Mannheim).The PCR reaction was run for 24 cycles (1 minute at 94° C., 1 minute at56° C., and 1 minute at 72° C.). An approximately 1.1 Kb band wasisolated on a low melt agarose gel electrophoresis and random primedusing the MEGAPRIME˜ Kit (Amersham) according to the manufacturer'sinstructions.

[0109] The filter was hybridized in a solution containing 6× SSC, 0.1%SDS, 5× Denhardt's, 200 μg/ml denatured, sheared salmon sperm DNA, and1×10⁵ cpm/ml of ³²P-labeled PCR fragment. The filter was hybridizedovernight at 65° C. The excess label was removed by two, 15 minutewashes with 2× SSC, 0.1% SDS at 65° C. The filter was exposed to filmovernight at −80° C. with two screens.

[0110] Eighteen positive colonies were detected. Six of these colonieswere cultured and subjected to a second round of filter lifts asdescribed above, and from this two positive clones were identified.Restriction endonuclease analysis showed that both contained anapproximate 2 Kb insert. One clone, designated M1.18.5.1, was sequenced,revealing a 2,170 bp coding region which contained regions of homologyto the protein tyrosine phosphatase family, especially IA-2/ICA512.Comparison of the full length human protein tyrosine phosphataseIA-2/ICA512 with M1.18.5.1 suggests that the coding region of M1.18.5.1is missing amino terminal sequence corresponding to approximately 400amino acids. The partial nucleic acid sequence and deduced amino acidsequence of M1.18.5.1 is shown in SEQ ID NO 1 and SEQ ID NO: 2.

[0111] M1.18.5.1 was re-transfected into COS-7 cells and assayed asdescribed above. In addition to the EmWi sera, the JoGr sera, which hada high titer to IA-2, was added to the screen and both detectedM1.18.5.1.

Example 5 Isolation of Human Islet Cell Antigen 1851

[0112] The 2,170 nucleotide sequence from M1.18.5.1 (SEQ ID NO 1) wasused to conduct a sequence search for a human homolog. A match was foundin the GenBank database (GenBank ID: TO361, clone ID: HFBCV88) submittedby The Institute of Genomic Research, Gaithersburg, Md., as an expressedsequence tag (EST) from a human fetal brain library (Stratagene CloningSystems). HFBCV88 (EST24415.seq), a 127 amino acid polypeptide, SEQ IDNO:5, had homology to a region of the cytoplasmic domain of M1.18.5.1.The closest human DNA sequence to HFBCV88 is HSICA512, islet cellantigen ICA-512.

[0113] An oligonucleotide primer (ZC10,011 SEQ ID NO:11) was made to aconserved region between 1851 and HFBCV88 which differed from thecorresponding sequence of mouse and human IA-2/ICA512 in that anarginine was substituted for a methionine. Combined with a 128 folddegenerate primer (ZC10,019 SEQ ID NO:14, AARGCNACNGTNGAYAAY, wherein Ris A or G, N is A, C, T, or G, and Y is C or T) which lies just upstreamof the transmembrane domain, in the extracellular domain, a portion ofthe human homologue of M1.18.5.1 was identified in human insulinoma cDNAby PCR. Briefly, a PCR reaction was performed in a 100 μl final volumeusing 12.5 ng Marathon-ready human insulinoma cDNA prepared according tomanufacturer's instruction (Marathon˜ cDNA Amplification Kit, Clontech),20 pmoles each of primers ZC 10,011 (SEQ ID NO:11) and ZC 10,019 (SEQ IDNO:14), and the reagents provided in the Marathon˜ PCR kit (Clontech)according to the manufacturer's instructions. The reaction was amplifiedfor 30 cycles (1 minute at 94° C., 30 seconds at 60° C., 5 minutes at68° C.) followed by a 10 minute extension at 72° C. An 800 bp (WK11111,SEQ ID NO:32) and a 1,200 bp (WK121315, SEQ ID NO:34) fragment wereisolated by low melt agarose gel electrophoresis.

[0114] A 3′RACE Marathon PCR was also performed in a 50 μl final volumeusing 12.5 ng Marathon-ready human insulinoma cDNA, 10 pmoles each ofprimers ZC 10,177 (SEQ ID NO:12) the complement to ZC 10,011, and AP-1(adaptor primer, supplied with kit), and the reagents provided in the3′RACE Marathon˜ PCR kit (Clontech), according to the manufacturer'sinstructions. The reaction was amplified for 30 cycles (30 seconds at94° C., 30 seconds at 68° C.). A 900 bp and a 2,000 bp (WK121111, SEQ IDNO:33) fragment were isolated by low melt agarose electrophoresis.

[0115] The 800 bp, (SEQ ID NO:32) 1,200 bp, (SEQ ID NO:34) and 2,000 bp(SEQ ID NO:33) PCR fragments were independently subcloned into pCR1(Invitrogen Inc., San Diego, Calif.), using the TA Cloning Kit(Invitrogen Inc.) according to the manufacturer's instructions. Theresulting plasmids (11.1.1, 11.1.2, and 11.1.3, respectfully) were usedto transform E. coli XL-1 cells. Transformants were screened forpresence of insert, followed by sequencing of the insert.

Example 6 Detection of Human Islet Cell Antigen Autoantibodies

[0116] An approximately 1.1 kb (SEQ ID NO:6) Eco RI-Hind III cytoplasmicfragment of human islet cell antigen 1851 cDNA was inserted into thevector pcDNAII (Invitrogen, San Diego, Calif.), and designated IL1851-3.The resultant polypeptide was transcribed and translated in vitro usinga TNT Coupled. Reticulocyte Lysate System (Promega), according themanufacturer's instructions.

[0117] The labeled, synthesized cytoplasmic portion of human islet cellantigen 1851 was used to screen diabetic sera from six patients, for thepresence of autoantibodies. Protein A-Sepharose immunoprecipitation, asdescribed above, showed that sera from all six reacted positively withthe in vitro synthesized, human islet cell antigen, and indicated thatthe major autoepitope is likely present on this polypeptide.

[0118] Additional immunoprecipitation assays were performed with aspectrum of serum samples, including 91 healthy control sera (median age22 years, range 1-49 years, 49% males and 51% females); 183 newlydiagnosed IDDM patients sampled at onset (median age 11 years, 51% malesand 49% females); and 60 first degree relatives of type I diabeticpatients sampled a mean of 2.0 years before onset (median age 12 years,58% males and 42% females). Parallel autoantibody assays used theintracellular domain of IA-2/ICA512. Immunoprecipitation assays were asdescribed above. Briefly, 4 μl of serum from diabetic or controlpatients were separately incubated in duplicate with 400 μl ³⁵Sradiolabeled antigen (cytoplasmic portion of human islet cell antigen1851, SEQ ID NO: 6, in immunoprecipitation buffer (10 mM Hepes, 0.05%BSA, 150 mM NaCl, 10 mM benzamidine, and 1% Triton X114)) at 4° C.overnight with mixing by gentle rotation (Hagopian et al., J. Clinc.Invest. 91:368-74, 1995). Antigen-antibody complexes were precipitatedusing 20 μl Protein A Sepharose, and the pellet was washed 3 times inice-cold wash buffer (which consisted of 10 mM HEPES pH 7.4, 150 mMNaCl, 0.25% BSA, and 0.25% Triton X-114) and one cold water wash.Antigen was dissociated from the pellet by boiling in the presence of 2%SDS and 5% β-mercaptoethanol, counted by scintillation counting inscintillation fluid, and the results expressed as islet cell antigen1851 index (Hagopian et al., Diabetes 42:631-36, 1993). Counts perminute reflect the level of autoantibodies present in the sera that cancapture the antigen. Assay cutoff was an index of 0.04, determined asthe mean +3 standard deviations of 91 control sera. Assay sensitivity,specificity, and positive predictive value were calculated (Hagopian etal., ibid., 1995).

[0119] Immunoprecipitation assays revealed autoantibodies in 56/183(30.6%) newly diagnosed IDDM patients, 28/60 (46.7%) first degreerelatives later progressing to clinical diabetes, but only 1/91 (1.1%)healthy control subject groups. For first degree relatives, thisrepresents a positive predictive value of 58% and a sensitivity of 48%.

[0120] Of sera from 153 newly diagnosed patients, 83 (54%) recognizedIC-2/ICA512 and 48 (31%) recognized islet cell antigen 1851. Only 1/48(2%) from the sera recognizing islet cell antigen 1851 did notprecipitate IA-2/ICA512, but 35/83 (42%) from the sera reactive withIA-2/ICA512 did not bind islet cell antigen 1851. Of those positive forboth antigens, reactivity to IA-2/ICA512 was generally stronger thanthat to islet cell antigen 1851.

[0121] The intracellular domains of human islet cell antigen 1851 andIA-2/ICA512 were expressed and radiolabeled by in vitro transcriptionand translation using a TNT Coupled Reticulocyte Lysate System(Promega), according the manufacturer's instructions, as describedabove. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andautoradiography of the resulting radiolabeled polypeptide revealed, forhuman islet cell antigen 1851, a major band of 46 kD and a minor band at33 kD, both immunoprecipitated by IDDM sera. Limited trypsin digest ofthe radiolabeled immunoprecipitated intracellular fragment of macaqueand human islet cell antigen 1851 and IA-2/ICA512 was done using themethod of Christie et al. (J. Exp. Med. 172:789-94, 1990), followed bySDS-PAGE and autoradiography, which revealed a 37 kD product from bothmacaque and human islet cell antigen 1851. This product was distinctfrom the 40 kD product produced by limited trypinization of theintracellular domain of IA-2/ICA512.

[0122] In order to test whether IA-2/ICA512 autoantibodies recognizedonly epitopes shared with islet cell antigen 1851, the intracellulardomain of IA-2/ICA512 was expressed in baby hamster kidney cells (BHKcells). The 1.2 kb IA-2/ICA512 intracellular fragment (SEQ ID NO:30)from Example 3 was ligated into pZEM219b under the SV40 promoter (Busbyet al., J. Biol. Chem. 266:15286-92, 1991) and cellular expression wasdetermined by immunocytochemistry using rabbit polyclonal antiserum toIA-2/ICA512 (Rabin et al., J. Immunol. 152:3183-88, 1994).IA-2/ICA512-transfected BHK cells were homogenized in homogenizationbuffer (0.25% Triton X-114, 10 mM benzamidine). Using Western blotting,the concentration of recombinant intracellular IA-2/ICA512 was estimatedat 7 μg/ml of cell extract.

[0123] Immunoprecipitation assays, as described above, were done usingradiolabeled islet cell antigen 1851 in the presence of 0.5 μg ofunlabeled IA-2/ICA512 per microliter of islet cell antigen 1851 positivesera, as a competitor. Islet cell antigen 1851 autoantibodies not fullyblocked by this amount of IA-2/ICA512 were subjected to repeatedimmunoprecipitation assays using a 2.5 fold increase of unlabeledIA-2/ICA512 as a competitor. As a control, extracts from non-transfectedBHK cells were used. Recombinant intracellular IA-2/ICA512 fully blockedislet cell antigen 1851 reactivity in 29/53 islet cell antigen 1851positive sera, while a median of 21.4% (range 3%-55%) of originalimmunoreactivity was retained in 24/53 sera. Increasing the IA-2/ICA512concentration did not reduce this residual immunoreactivity, suggestingthat unique islet cell antigen 1851 epitopes are being recognized incertain sera.

Example 7 Cloning the Remaining 5′ Sequence of Macaque and Human IsletCell Antigen 1851 cDNA

[0124] To obtain the remaining 5′ macaque cDNA sequence one pool (#12)from the macaque library described in Example 1 was plated at 10,000colonies/150 mm plate. Filter lifts were prepared (Maniatis et al.(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1982)and denatured with 0.5 M NaOH for four minutes, neutralized with 1 MTris pH 8.0 for 2 minutes followed by renaturation with 1 M Tris pH8.0/1.5 M NaCl for 2 minutes. Filters were cross linked in a UVStratalinker (1200 μJ) (Stratagene Cloning Systems, La Jolla, Calif.).The filters were prehybridized in 20 ml hybridization buffer (6× SSC,0.5% SDS, 5× Denhardts and 0.2 mg/ml boiled salmon sperm DNA) overnightat 65° C. The filters were then hybridized in 20 ml hybridization buffercontaining 1×10⁶ cpm/ml ^(γ32)P-ATP labeled hybridization probe (ZC10504SEQ ID NO:18) overnight at 65° C. The labeled hybridization probe wasprepared by adding to a 5 μl final volume 30 pmol oligo ZC10504 (SEQ IDNO:18), T4 polynucleotide kinase buffer, 37.5 pmol ^(γ32)P-ATP and 10 UT4 polynucleotide kinase The reaction was incubated for 1 hour at roomtemperature and unincorporated ATP was removed using a Stratagene pushcolumn according the manufacturer's instructions (Stratagene CloningSystems, La Jolla, Calif.). Following the hybridization, excess unboundlabel was removed from the filters with eight washes in 2× SSC/0.1 SDS(2 times with 20 ml, 5 times with 30 ml and a final wash in 100 ml) for5 to 10 minutes at 65° C. The filters were exposed to film overnight at−80° C.

[0125] Several positive colonies were detected. One of these colonieswas cultured from a replica plated colony and subjected to sequenceanalysis. The clone, 12.10504.1, contained 2,736 bp coding region (SEQID NO:23), containing the cytoplasmic and transmembrane domains andextending the 5′ end of the macaque extracellular domain sequence (SEQID NO:1) by 609 bp.

[0126] 5′ RACE PCR was used to generate the remaining 5′ cDNA fragmentsof macaque islet cell antigen 1851. To a 50 μl final volume was added 5pmol of a vector-specific oligonucleotide primer (ZC11197, SEQ IDNO:29), 5 pmol of a macaque specific primer (ZC11654, SEQ ID NO:28), 1ng macaque islet cell cDNA library from Example 1, 40 mM dNTPs, TAQPolymerase buffer and 1.25 U TAQ Polymerase. A one minute denaturationat 94° C. was followed by 30 amplification cycles (30 seconds at 94° C.,1 minute at 60° C., 2 minutes at 72° C.) followed by a 6 minuteextension at 72° C.).

[0127] Four independent 5′RACE PCR reactions were run, each using adifferent pool from the macaque library as template. Four fragments wereobtained, a 738 bp fragment (SEQ. ID. No. 24) extending the 5′ end by246 bp; a 932 bp fragment (SEQ ID NO:25) extending the 5′ end by 193 bp;a 999 bp fragment (SEQ ID NO:26) extending the 5′ end by 68 bp and a1011 bp fragment (SEQ ID NO:27) which contained the remaining 5′sequence with the exception of the start methionine. The fragments wereisolated by agarose electrophoresis, excised and separated from theagarose using the Qiagen Qiaquick Gel Extraction System (Qiagen, Inc.,Chatsworth, Calif.) according to manufacturer's instruction. Thefragments were subcloned into pGEM-T (Promega Corp., Madison, Wis.),using the TA Cloning Kit (Promega Corp.) according to the manufacturer'sinstructions. The resulting plasmids pJML8, 7, 9 and 10 respectfully,were used to transform E. coli DH10B cells. Transformants were screenedfor presence of insert, followed by sequencing of the insert.

[0128] The 5′ RACE fragments (SEQ ID Nos:23, 24, 25, 26 and 27) containoverlapping segments and were aligned with the macaque islet cellantigen 1851 sequence of SEQ ID NO:1 to give a full length macaque isletcell antigen 1851 DNA sequence as represented in SEQ ID NO:15.Comparison of the human protein tyrosine phosphatase IA-2/ICA512 cDNAand amino acid sequences with those of the macaque islet cell antigen1851 cDNA and amino acid sequences (SEQ ID NOs: 15 and 16) suggests thatthe coding region is missing the start methionine.

[0129] A vector containing the full length macaque sequence can becreated using PCR. The macaque 5′ RACE fragments (SEQ ID NOs: 23, 24,25, 26 and 27) can be joined using PCR. A clone shown to possess thecomplete coding sequence can then be digested with convenientrestriction sites and subcloned into a vector of choice. Clones can bescreened for correct insertion of the full length sequence and subjectedto DNA sequence analysis.

[0130] PCR using macaque derived primers was done to identify remaining5′ cDNA sequence for the human islet cell antigen 1851 (SEQ ID NO:6). Toa 50 μl final volume was added 5 pmol each of two gene-specificoligonucleotide primers ZC10504, SEQ ID NO:18 and ZC11653, SEQ ID NO:17,1 ng Marathon-ready insulinoma cDNA, prepared according tomanufacturer's instruction (Marathon˜ cDNA Amplification Kit, Clontech),40 mM dNTPs, TAQ Polymerase buffer and 1.25 U TAQ Polymerase. Thereaction was denatured at 94° C. for one minute, amplified for 30 cycles(30 seconds at 94° C., 1 minute at 63° C., 2 minutes at 72° C.),followed by a 6 minute extension at 72° C.).

[0131] A 1263 bp fragment (SEQ ID NO:31) was isolated by agaroseelectrophoresis. The isolated fragment was then excised and subclonedinto pGEM-T using the TA Cloning Kit (Promega, Corp.), as describedabove. The clones were then analyzed for the presence of insert, andthose containing insert were subjected to DNA sequence analysis. Thehuman islet cell antigen 1851 fragments can be joined using PCR to givethe human sequence as represented in SEQ ID NO:21. Clones can bescreened for correct insertion of the fragments and subjected to DNAsequence analysis. Comparison of the human protein tyrosine phosphataseIA-2/ICA512 cDNA with that of the human islet cell antigen 1851sequences (SEQ ID NO:21 and 22) suggests that the coding region ismissing 5′ sequence corresponding to approximately 600 bp. Including the3′ untranslated region, but not the 5′ untranslated region, theestimated mRNA size for the human sequence is 5 kb, which is consistentwith the 5.5 kb mRNA observed in Northern blots discussed below. Toobtain the remaining 5′ human islet cell antigen 1851 cDNA sequence,additional PCR or 5′ RACE PCR reactions can be performed as describedabove.

Example 8 Tissue Distribution

[0132] Human Multiple Tissue Northern Blots (MTN I, MTN II, and MTN III;Clontech, Palo Alto, Calif.) were probed to determine the tissuedistribution of human islet cell antigen 1851 expression. A 38nucleotide oligonucleotide sequence just external to the transmembraneregion of human islet cell antigen 1851, which is distinct from thecorresponding sequence of IA-2/ICA512 (SEQ ID NO:18) was radioactivelylabeled with γ³²P using a T4 nucleotide kinase (GIBCO BRL, Gaithersburg,Md.) according to the manufacturer's specifications. ExpressHyb˜(Clontech) solution was used for prehybridization and as a hybridizingsolution for the Northern blots. Hybridization took place overnight at37° C. using 5×10⁶ cpm/ml of labeled probe. The blots were then washedthree times at room temperature, once at 50° C. for 30 minutes, once at60° C., in 6× SSC, 0.1% SDS. A final wash at 68° C. with 2× SSC, 0.05%SDS for 20 minutes was done prior to autoradiography. Two transcriptsizes were detected. A strong 5.5 kb band and a weaker 3.3 kb band weredetected in brain, pancreas and prostate, with lesser signals in spinalcord, thyroid, adrenal and GI tract. With the exception of prostate,this represents the expected neuroendocrine distribution.

[0133] In order to define tissue localization further, in situhybridization was performed on macaque pancreas, adrenal gland andmuscle. The 38 nucleotide islet cell antigen 1851 oligonucleotide (SEQID NO:18), a 38 bp IC-2/ICA512 oligonucleotide (SEQ ID NO:19) and a 30bp insulin β-chain probe for pancreatic islets (Petersen et al.,Diabetes 42:484-95, 1993) (SEQ ID NO:20) were end-labeled with ³³P-dATP(New England Nuclear, Boston, Mass.) using terminal deoxytransferase(GIBCO BRL) according to manufacturer's instructions. Frozen sections(14 μm) from macaque pancreas, adrenal, pituitary and muscle were fixedin 4% paraformaldehyde, followed by acetylation with acetic anhydrideand then delipidated in chloroform prior to use. Labeled probes (2pmol/ml) were incubated on the sections overnight and then washed in twochanges of 1× SSC at 60° C. for 30 minutes, followed by dehydration inethanol and apposition to autoradiography film (Hyperfilm Betamax,Amersham Corp., Arlington Heights, Ill.) for 2 to 6 days. The slideswere then coated with NTB2 Track emulsion (Eastman Kodak, Rochester,N.Y.) and exposed for 12-18 days before development and counterstainwith cresyl violet. Images were captured using a Dage 72 CCD camera anda MCID M2 imaging system (Imaging Research, Ontario, Canada). Stronghybridization was detected in pancreatic islets and adrenal medulla butnot in muscle. The IA-2/ICA512 and the insulin β chain probes hybridizedto islets.

[0134] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention. Accordingly, theinvention is not limited except as by the appended claims.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:34 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 2171 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: Coding Sequence (B) LOCATION: 1...1923 (D) OTHER INFORMATION:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAA TTC GGC ACG AGC GGA GTT CAGGAC GAC GAT GAC AGA CTT TAC CAA 48 Glu Phe Gly Thr Ser Gly Val Gln AspAsp Asp Asp Arg Leu Tyr Gln 1 5 10 15 GAG GTC CAT CGT CTG AGT GCC ACACTC GGG GGC CTC CTG CAG GAC CAC 96 Glu Val His Arg Leu Ser Ala Thr LeuGly Gly Leu Leu Gln Asp His 20 25 30 GGG TCT CGA CTC TCG CCT GGA GCC CTCCCC TTT GCA AAG CCC CTC AAA 144 Gly Ser Arg Leu Ser Pro Gly Ala Leu ProPhe Ala Lys Pro Leu Lys 35 40 45 ATG GAG AGG AAG AAA TCC GAG CGC CCT GAGGCT TCC CTG TCT TCA GAA 192 Met Glu Arg Lys Lys Ser Glu Arg Pro Glu AlaSer Leu Ser Ser Glu 50 55 60 GAG GAG ACT GCC GGA GTG GAG AAC GTC AAG AGCCAG ACG TAT TCC AAA 240 Glu Glu Thr Ala Gly Val Glu Asn Val Lys Ser GlnThr Tyr Ser Lys 65 70 75 80 GAC CTG CTG GGG CAG CAG CCG CAT TCG GAG CCCGGG GCA GGC GCG TTT 288 Asp Leu Leu Gly Gln Gln Pro His Ser Glu Pro GlyAla Gly Ala Phe 85 90 95 GGG GAG CTC CAA AAC CAG ATG CCT GGG CCC TCG GAGGAG GAG CAG AGC 336 Gly Glu Leu Gln Asn Gln Met Pro Gly Pro Ser Glu GluGlu Gln Ser 100 105 110 CTT CCA GCG GGT GCT CAG GAG GCC CTC GGC GAC GGCCTG CAA TTG GAA 384 Leu Pro Ala Gly Ala Gln Glu Ala Leu Gly Asp Gly LeuGln Leu Glu 115 120 125 GTC AAG CCT TCC GAG GAA GAG GCA CGG TGC TAC ATCGTG ACA GAC AGA 432 Val Lys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile ValThr Asp Arg 130 135 140 GAC CCC CTG CGC CCC GAG GAA GGA AGG CAG CTG GTGGAG GAC GTC GCC 480 Asp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val GluAsp Val Ala 145 150 155 160 CGC CTC CTG CAG ATG CCC AGC AGC ACA TTC GCCGAC GTG GAG GTT CTC 528 Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala AspVal Glu Val Leu 165 170 175 GGA CCA GCA GTG ACC TTC AAA GTG GGC GCC AATGTC CAG AAC GTG ACC 576 Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn ValGln Asn Val Thr 180 185 190 ACT GCG GAT GTG GAG AAG GCC ACA GTT GAC AACAAA GAC AAA CTG GAG 624 Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn LysAsp Lys Leu Glu 195 200 205 GAA ACC TCT GGA CTG AAA ATT CTT CAA ACC GGAGTC GGG TCG AAA AGC 672 Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly ValGly Ser Lys Ser 210 215 220 AAA CTC AAG TTC CTG CCT CCT CAG GCG GAG CAAGAA GAC TCA ACC AAG 720 Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln GluAsp Ser Thr Lys 225 230 235 240 TTC ATC GCG CTC ACC CTG GTC TCC CTC GCCTGC ATC CTG GGC GTC CTC 768 Phe Ile Ala Leu Thr Leu Val Ser Leu Ala CysIle Leu Gly Val Leu 245 250 255 CTG GCC TCT GGC CTC ATC TAC TGC CTA CGCCAT AGC TCT CAG CAC AGG 816 Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg HisSer Ser Gln His Arg 260 265 270 CTG AAG GAG AAG CTC TCG GGA CTA GGG CGCGAC CCA GGT GCA GAT GCC 864 Leu Lys Glu Lys Leu Ser Gly Leu Gly Arg AspPro Gly Ala Asp Ala 275 280 285 ACC GCC GCC TAC CAG GAG CTG TGC CGC CAGCGT ATG GCC ACG CGG CCA 912 Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln ArgMet Ala Thr Arg Pro 290 295 300 CCA GAC CGG CCC GAG GGC CCG CAC ACA TCCCGC ATC AGC AGC GTC TCG 960 Pro Asp Arg Pro Glu Gly Pro His Thr Ser ArgIle Ser Ser Val Ser 305 310 315 320 TCC CAG TTC AGC GAC GGG CCG ATG CCCAGC CCC TCC GCA CGC AGC AGC 1008 Ser Gln Phe Ser Asp Gly Pro Met Pro SerPro Ser Ala Arg Ser Ser 325 330 335 GCC TCG TCC TGG TCC GAG GAG CCC GTGCAG TCC AAC ATG GAC ATC TCC 1056 Ala Ser Ser Trp Ser Glu Glu Pro Val GlnSer Asn Met Asp Ile Ser 340 345 350 ACC GGC CAC ATG ATC CTG TCC TAC ATGGAG GAC CAC CTG AAG AAC AAG 1104 Thr Gly His Met Ile Leu Ser Tyr Met GluAsp His Leu Lys Asn Lys 355 360 365 AAC CGG CTG GAG AAG GAG TGG GAG GCGCTG TGT GCC TAC CAG GCG GAG 1152 Asn Arg Leu Glu Lys Glu Trp Glu Ala LeuCys Ala Tyr Gln Ala Glu 370 375 380 CCC AAC AGC TCA CTT GTG GCC CAG AAGGAG GAG AAT GTG CCC AAG AAC 1200 Pro Asn Ser Ser Leu Val Ala Gln Lys GluGlu Asn Val Pro Lys Asn 385 390 395 400 CGC TCC CTG GCC GTG CTG ACC TATGAC CAC TCC CGG GTC CTA CTG AAG 1248 Arg Ser Leu Ala Val Leu Thr Tyr AspHis Ser Arg Val Leu Leu Lys 405 410 415 GCG GAG AAC AGC CAC AGC CAC TCGGAC TAC ATC AAC GCC AGC CCC ATC 1296 Ala Glu Asn Ser His Ser His Ser AspTyr Ile Asn Ala Ser Pro Ile 420 425 430 ATG GAT CAC GAC CCG AGG AAC CCCGCG TAC ATC GCC ACC CAG GGA CCG 1344 Met Asp His Asp Pro Arg Asn Pro AlaTyr Ile Ala Thr Gln Gly Pro 435 440 445 CTG CCC GCC ACC GTG GCC GAC TTTTGG CAG ATG GTG TGG GAG AGC GGC 1392 Leu Pro Ala Thr Val Ala Asp Phe TrpGln Met Val Trp Glu Ser Gly 450 455 460 TGC GTG GTG ATC GTC ATG CTG ACACCC CTC ACA GAG AAC GGC GTC CGG 1440 Cys Val Val Ile Val Met Leu Thr ProLeu Thr Glu Asn Gly Val Arg 465 470 475 480 CAG TGC TAC CAC TAC TGG CCAGAT GAA GGC TCC AAC CTC TAC CAC ATC 1488 Gln Cys Tyr His Tyr Trp Pro AspGlu Gly Ser Asn Leu Tyr His Ile 485 490 495 TAT GAG GTG AAC CTG GTC TCCGAG CAC ATC TGG TGC GAG GAC TTT CTG 1536 Tyr Glu Val Asn Leu Val Ser GluHis Ile Trp Cys Glu Asp Phe Leu 500 505 510 GTG AGG AGC TTC TAT CTG AAGAAC CTG CAG ACC AAC GAG ACG CGC ACC 1584 Val Arg Ser Phe Tyr Leu Lys AsnLeu Gln Thr Asn Glu Thr Arg Thr 515 520 525 GTG ACC CAG TTC CAC TTC CTGAGT TGG TAT GAC CGA GGA GTC CCC TCC 1632 Val Thr Gln Phe His Phe Leu SerTrp Tyr Asp Arg Gly Val Pro Ser 530 535 540 TCC TCA AGA TCC CTC CTG GACTTC CGC AGA AAA GTA AAC AAG TGC TAC 1680 Ser Ser Arg Ser Leu Leu Asp PheArg Arg Lys Val Asn Lys Cys Tyr 545 550 555 560 AGG GGC CGT TCT TGT CCAATA ATT GTT CAT TGC AGT GAC GGT GCA GGC 1728 Arg Gly Arg Ser Cys Pro IleIle Val His Cys Ser Asp Gly Ala Gly 565 570 575 CGG AGC GGC ACC TAC GTCCTG ATC GAC ATG GTT CTC AAC AAG ATG GCC 1776 Arg Ser Gly Thr Tyr Val LeuIle Asp Met Val Leu Asn Lys Met Ala 580 585 590 AAA GGT GCT AAA GAG ATTGAT ATC GCA GCA ACC CTG GAG CAC TTG AGG 1824 Lys Gly Ala Lys Glu Ile AspIle Ala Ala Thr Leu Glu His Leu Arg 595 600 605 GAC CAG AGA CCC GGC ATGGTC CAG ACG AAG GAG CAG TTT GAG TTC GCG 1872 Asp Gln Arg Pro Gly Met ValGln Thr Lys Glu Gln Phe Glu Phe Ala 610 615 620 CTG ACA GCC GTG GCT GAAGAG GTG AAT GCC ATC CTC AAG GCC CTT CCC 1920 Leu Thr Ala Val Ala Glu GluVal Asn Ala Ile Leu Lys Ala Leu Pro 625 630 635 640 CAG TGAGCAGCGGCCTCGGGGCC TCGGGGGAGC CCCCACCCCC CGGATGTCGT CAGGAA 1979 Gln TCGTGATCTGACTTTAATTG TGTGTCTTCT ATTATAACTG CATAGTAATA GGGCCCTTAG 2039 CTCTCCCGTAGTCAGCGCAG TTTAGCAGTT AAGCAGTTAA AATGTGTATT TTTGTTTAAT 2099 CCAACAATAATAAAGAGAGA TTTGTGGAAA AATCCCAAAA AAAAAAAAAA AAAAAAAAAA 2159 AAAAAACTCGAG 2171 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 641 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Glu Phe Gly ThrSer Gly Val Gln Asp Asp Asp Asp Arg Leu Tyr Gln 1 5 10 15 Glu Val HisArg Leu Ser Ala Thr Leu Gly Gly Leu Leu Gln Asp His 20 25 30 Gly Ser ArgLeu Ser Pro Gly Ala Leu Pro Phe Ala Lys Pro Leu Lys 35 40 45 Met Glu ArgLys Lys Ser Glu Arg Pro Glu Ala Ser Leu Ser Ser Glu 50 55 60 Glu Glu ThrAla Gly Val Glu Asn Val Lys Ser Gln Thr Tyr Ser Lys 65 70 75 80 Asp LeuLeu Gly Gln Gln Pro His Ser Glu Pro Gly Ala Gly Ala Phe 85 90 95 Gly GluLeu Gln Asn Gln Met Pro Gly Pro Ser Glu Glu Glu Gln Ser 100 105 110 LeuPro Ala Gly Ala Gln Glu Ala Leu Gly Asp Gly Leu Gln Leu Glu 115 120 125Val Lys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile Val Thr Asp Arg 130 135140 Asp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val Glu Asp Val Ala 145150 155 160 Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp Val Glu ValLeu 165 170 175 Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn Val Gln AsnVal Thr 180 185 190 Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn Lys AspLys Leu Glu 195 200 205 Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly ValGly Ser Lys Ser 210 215 220 Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu GlnGlu Asp Ser Thr Lys 225 230 235 240 Phe Ile Ala Leu Thr Leu Val Ser LeuAla Cys Ile Leu Gly Val Leu 245 250 255 Leu Ala Ser Gly Leu Ile Tyr CysLeu Arg His Ser Ser Gln His Arg 260 265 270 Leu Lys Glu Lys Leu Ser GlyLeu Gly Arg Asp Pro Gly Ala Asp Ala 275 280 285 Thr Ala Ala Tyr Gln GluLeu Cys Arg Gln Arg Met Ala Thr Arg Pro 290 295 300 Pro Asp Arg Pro GluGly Pro His Thr Ser Arg Ile Ser Ser Val Ser 305 310 315 320 Ser Gln PheSer Asp Gly Pro Met Pro Ser Pro Ser Ala Arg Ser Ser 325 330 335 Ala SerSer Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile Ser 340 345 350 ThrGly His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn Lys 355 360 365Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala Glu 370 375380 Pro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn Val Pro Lys Asn 385390 395 400 Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu LeuLys 405 410 415 Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala SerPro Ile 420 425 430 Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala ThrGln Gly Pro 435 440 445 Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met ValTrp Glu Ser Gly 450 455 460 Cys Val Val Ile Val Met Leu Thr Pro Leu ThrGlu Asn Gly Val Arg 465 470 475 480 Gln Cys Tyr His Tyr Trp Pro Asp GluGly Ser Asn Leu Tyr His Ile 485 490 495 Tyr Glu Val Asn Leu Val Ser GluHis Ile Trp Cys Glu Asp Phe Leu 500 505 510 Val Arg Ser Phe Tyr Leu LysAsn Leu Gln Thr Asn Glu Thr Arg Thr 515 520 525 Val Thr Gln Phe His PheLeu Ser Trp Tyr Asp Arg Gly Val Pro Ser 530 535 540 Ser Ser Arg Ser LeuLeu Asp Phe Arg Arg Lys Val Asn Lys Cys Tyr 545 550 555 560 Arg Gly ArgSer Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala Gly 565 570 575 Arg SerGly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met Ala 580 585 590 LysGly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu Arg 595 600 605Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe Ala 610 615620 Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu Pro 625630 635 640 Gln (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 894 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION: 1...894 (D) OTHERINFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CGC CAT AGC TCTCAG CAC AGG CTG AAG GAG AAG CTC TCG GGA CTA GGG 48 Arg His Ser Ser GlnHis Arg Leu Lys Glu Lys Leu Ser Gly Leu Gly 1 5 10 15 GGC GAC CCA GGTGCA GAT GCC ACT GCC GCC TAC CAG GAG CTG TGC CGC 96 Gly Asp Pro Gly AlaAsp Ala Thr Ala Ala Tyr Gln Glu Leu Cys Arg 20 25 30 CAG CGT ATG GCC ACGCGG CCA CCA GAC CGA CCT GAG GGC CCG CAC ACG 144 Gln Arg Met Ala Thr ArgPro Pro Asp Arg Pro Glu Gly Pro His Thr 35 40 45 TCA CGC ATC AGC AGC GTCTCA TCC CAG TTC AGC GAC GGG CCG ATC CCC 192 Ser Arg Ile Ser Ser Val SerSer Gln Phe Ser Asp Gly Pro Ile Pro 50 55 60 AGC CCC TCC GCA CGC AGC AGCGCC TCA TCC TGG TCC GAG GAG CCT GTG 240 Ser Pro Ser Ala Arg Ser Ser AlaSer Ser Trp Ser Glu Glu Pro Val 65 70 75 80 CAG TCC AAC ATG GAC ATC TCCACC GGC CAC ATG ATC CTG TCC TAC ATG 288 Gln Ser Asn Met Asp Ile Ser ThrGly His Met Ile Leu Ser Tyr Met 85 90 95 GAG GAC CAC CTG AAG AAC AAG AACCGG CTG GAG AAG GAG TGG GAA GCG 336 Glu Asp His Leu Lys Asn Lys Asn ArgLeu Glu Lys Glu Trp Glu Ala 100 105 110 CTG TGC GCC TAC CAG GCG GAG CCCAAC AGC TCG TTC GTG GCC CAG AGG 384 Leu Cys Ala Tyr Gln Ala Glu Pro AsnSer Ser Phe Val Ala Gln Arg 115 120 125 GAG GAG AAC GTG CCC AAG AAC CGCTCC CTG GCC GTG CTG ACC TAT GAC 432 Glu Glu Asn Val Pro Lys Asn Arg SerLeu Ala Val Leu Thr Tyr Asp 130 135 140 CAC TCC CGG GTC CTG CTG AAG GCGGAG AAC AGC CAC AGC CAC TCA GAC 480 His Ser Arg Val Leu Leu Lys Ala GluAsn Ser His Ser His Ser Asp 145 150 155 160 TAC ATC AAC GCT AGC CCC ATCATG GAT CAC GAC CCG AGG AAC CCC GCG 528 Tyr Ile Asn Ala Ser Pro Ile MetAsp His Asp Pro Arg Asn Pro Ala 165 170 175 TAC ATC GCC ACC CAG GGA CCGCTG CCC GCC ACC GTG GCT GAC TTT TGG 576 Tyr Ile Ala Thr Gln Gly Pro LeuPro Ala Thr Val Ala Asp Phe Trp 180 185 190 CAG ATG GTG TGG GAG AGC GGCTGC GTG GTG ATC GTC ATG CTG ACA CCC 624 Gln Met Val Trp Glu Ser Gly CysVal Val Ile Val Met Leu Thr Pro 195 200 205 CTC GCG GAG AAC GGC GTC CGGCAG TGC TAC CAC TAC TGG CCG GAT GAA 672 Leu Ala Glu Asn Gly Val Arg GlnCys Tyr His Tyr Trp Pro Asp Glu 210 215 220 GGC TCC AAT CTC TAC CAC ATCTAT GAG GTG AAC CTG GTC TCC GAG CAC 720 Gly Ser Asn Leu Tyr His Ile TyrGlu Val Asn Leu Val Ser Glu His 225 230 235 240 ATC TGG TGT GAG GAC TTCCTG GTG AGG AGC TTC TAT CTG AAG AAC CTG 768 Ile Trp Cys Glu Asp Phe LeuVal Arg Ser Phe Tyr Leu Lys Asn Leu 245 250 255 CAG ACC AAC GAG ACG CGCACC GTG ACG CAG TTC CAC TTC CTG AGT TGG 816 Gln Thr Asn Glu Thr Arg ThrVal Thr Gln Phe His Phe Leu Ser Trp 260 265 270 TAT GAC CGA GGA GTC CCTTCC TCC TCA AGG TCC CTC CTG GAC TTC CGC 864 Tyr Asp Arg Gly Val Pro SerSer Ser Arg Ser Leu Leu Asp Phe Arg 275 280 285 AGA AAA GTA AAC AAG TGCTAC AGG GGC CGT 894 Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg 290 295 (2)INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:298 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Arg His Ser Ser Gln His Arg LeuLys Glu Lys Leu Ser Gly Leu Gly 1 5 10 15 Gly Asp Pro Gly Ala Asp AlaThr Ala Ala Tyr Gln Glu Leu Cys Arg 20 25 30 Gln Arg Met Ala Thr Arg ProPro Asp Arg Pro Glu Gly Pro His Thr 35 40 45 Ser Arg Ile Ser Ser Val SerSer Gln Phe Ser Asp Gly Pro Ile Pro 50 55 60 Ser Pro Ser Ala Arg Ser SerAla Ser Ser Trp Ser Glu Glu Pro Val 65 70 75 80 Gln Ser Asn Met Asp IleSer Thr Gly His Met Ile Leu Ser Tyr Met 85 90 95 Glu Asp His Leu Lys AsnLys Asn Arg Leu Glu Lys Glu Trp Glu Ala 100 105 110 Leu Cys Ala Tyr GlnAla Glu Pro Asn Ser Ser Phe Val Ala Gln Arg 115 120 125 Glu Glu Asn ValPro Lys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp 130 135 140 His Ser ArgVal Leu Leu Lys Ala Glu Asn Ser His Ser His Ser Asp 145 150 155 160 TyrIle Asn Ala Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala 165 170 175Tyr Ile Ala Thr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp 180 185190 Gln Met Val Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro 195200 205 Leu Ala Glu Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu210 215 220 Gly Ser Asn Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser GluHis 225 230 235 240 Ile Trp Cys Glu Asp Phe Leu Val Arg Ser Phe Tyr LeuLys Asn Leu 245 250 255 Gln Thr Asn Glu Thr Arg Thr Val Thr Gln Phe HisPhe Leu Ser Trp 260 265 270 Tyr Asp Arg Gly Val Pro Ser Ser Ser Arg SerLeu Leu Asp Phe Arg 275 280 285 Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg290 295 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 127 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 5: Ala Ser Pro Ile Met Asp His Asp Pro Arg AsnPro Ala Tyr Ile Ala 1 5 10 15 Thr Gln Gly Pro Leu Pro Ala Thr Val AlaAsp Phe Trp Gln Met Val 20 25 30 Trp Glu Ser Gly Cys Val Val Ile Val MetLeu Thr Pro Leu Ala Glu 35 40 45 Asn Gly Val Arg Gln Cys Tyr His Tyr TrpPro Asp Glu Gly Ser Asn 50 55 60 Leu Tyr His Ile Tyr Glu Val Asn Leu ValSer Glu His Ile Trp Cys 65 70 75 80 Glu Asp Phe Leu Val Arg Ser Phe TyrLeu Lys Asn Leu Gln Thr Asn 85 90 95 Glu Thr Arg Thr Val Thr Gln Phe ProLeu Ser Xaa Trp Tyr Asp Arg 100 105 110 Xaa Val Pro Ser Phe Leu Lys ValPro Xaa Trp Thr Ser Ala Glu 115 120 125 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1163 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (ix) FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION:27...1154 (D) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AAGCTATGCA TCAAGCTTCC ACCATG CGC CAT AGC TCT CAG CAC AGG CTG AAG 53Arg His Ser Ser Gln His Arg Leu Lys 1 5 GAG AAG CTC TCG GGA CTA GGG GGCGAC CCA GGT GCA GAT GCC ACT GCC 101 Glu Lys Leu Ser Gly Leu Gly Gly AspPro Gly Ala Asp Ala Thr Ala 10 15 20 25 GCC TAC CAG GAG CTG TGC CGC CAGCGT ATG GCC ACG CGG CCA CCA GAC 149 Ala Tyr Gln Glu Leu Cys Arg Gln ArgMet Ala Thr Arg Pro Pro Asp 30 35 40 CGA CCT GAG GGC CCG CAC ACG TCA CGCATC AGC AGC GTC TCA TCC CAG 197 Arg Pro Glu Gly Pro His Thr Ser Arg IleSer Ser Val Ser Ser Gln 45 50 55 TTC AGC GAC GGG CCG ATC CCC AGC CCC TCCGCA CGC AGC AGC GCC TCA 245 Phe Ser Asp Gly Pro Ile Pro Ser Pro Ser AlaArg Ser Ser Ala Ser 60 65 70 TCC TGG TCC GAG GAG CCT GTG CAG TCC AAC ATGGAC ATC TCC ACC GGC 293 Ser Trp Ser Glu Glu Pro Val Gln Ser Asn Met AspIle Ser Thr Gly 75 80 85 CAC ATG ATC CTG TCC TAC ATG GAG GAC CAC CTG AAGAAC AAG AAC CGG 341 His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys AsnLys Asn Arg 90 95 100 105 CTG GAG AAG GAG TGG GAA GCG CTG TGC GCC TACCAG GCG GAG CCC AAC 389 Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr GlnAla Glu Pro Asn 110 115 120 AGC TCG TTC GTG GCC CAG AGG GAG GAG AAC GTGCCC AAG AAC CGC TCC 437 Ser Ser Phe Val Ala Gln Arg Glu Glu Asn Val ProLys Asn Arg Ser 125 130 135 CTG GCC GTG CTG ACC TAT GAC CAC TCC CGG GTCCTG CTG AAG GCG GAG 485 Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val LeuLeu Lys Ala Glu 140 145 150 AAC AGC CAC AGC CAC TCA GAC TAC ATC AAC GCTAGC CCC ATC ATG GAT 533 Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala SerPro Ile Met Asp 155 160 165 CAC GAC CCG AGG AAC CCC GCG TAC ATC GCC ACCCAG GGA CCG CTG CCC 581 His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr GlnGly Pro Leu Pro 170 175 180 185 GCC ACC GTG GCT GAC TTT TGG CAG ATG GTGTGG GAG AGC GGC TGC GTG 629 Ala Thr Val Ala Asp Phe Trp Gln Met Val TrpGlu Ser Gly Cys Val 190 195 200 GTG ATC GTC ATG CTG ACA CCC CTC GCG GAGAAC GGC GTC CGG CAG TGC 677 Val Ile Val Met Leu Thr Pro Leu Ala Glu AsnGly Val Arg Gln Cys 205 210 215 TAC CAC TAC TGG CCG GAT GAA GGC TCC AATCTC TAC CAC ATC TAT GAG 725 Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn LeuTyr His Ile Tyr Glu 220 225 230 GTG AAC CTG GTC TCC GAG CAC ATC TGG TGTGAG GAC TTC CTG GTG AGG 773 Val Asn Leu Val Ser Glu His Ile Trp Cys GluAsp Phe Leu Val Arg 235 240 245 AGC TTC TAT CTG AAG AAC CTG CAG ACC AACGAG ACG CGC ACC GTG ACG 821 Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn GluThr Arg Thr Val Thr 250 255 260 265 CAG TTC CAC TTC CTG AGT TGG TAT GACCGA GGA GTC CCT TCC TCC TCA 869 Gln Phe His Phe Leu Ser Trp Tyr Asp ArgGly Val Pro Ser Ser Ser 270 275 280 AGG TCC CTC CTG GAC TTC CGC AGA AAAGTA AAC AAG TGC TAC AGG GGC 917 Arg Ser Leu Leu Asp Phe Arg Arg Lys ValAsn Lys Cys Tyr Arg Gly 285 290 295 CGT TCT TGT CCA ATA ATT GTT CAT TGCAGT GAC GGT GCA GGC CGG AGC 965 Arg Ser Cys Pro Ile Ile Val His Cys SerAsp Gly Ala Gly Arg Ser 300 305 310 GGC ACC TAC GTC CTG ATC GAC ATG GTTCTC AAC AAG ATG GCC AAA GGT 1013 Gly Thr Tyr Val Leu Ile Asp Met Val LeuAsn Lys Met Ala Lys Gly 315 320 325 GCT AAA GAG ATT GAT ATC GCA GCG ACCCTG GAG CAC TTG AGG GAC CAG 1061 Ala Lys Glu Ile Asp Ile Ala Ala Thr LeuGlu His Leu Arg Asp Gln 330 335 340 345 AGA CCC GGC ATG GTC CAG ACG AAGGAG CAG TTT GAG TTC GCG CTG ACA 1109 Arg Pro Gly Met Val Gln Thr Lys GluGln Phe Glu Phe Ala Leu Thr 350 355 360 GCC GTG GCT GAG GAG GTG AAC GCCATC CTC AAG GCC CTG CCC CAG TGAGA 1159 Ala Val Ala Glu Glu Val Asn AlaIle Leu Lys Ala Leu Pro Gln 365 370 375 ATTC 1163 (2) INFORMATION FORSEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 376 amino acids(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 7: Arg His Ser Ser Gln His Arg Leu Lys Glu LysLeu Ser Gly Leu Gly 1 5 10 15 Gly Asp Pro Gly Ala Asp Ala Thr Ala AlaTyr Gln Glu Leu Cys Arg 20 25 30 Gln Arg Met Ala Thr Arg Pro Pro Asp ArgPro Glu Gly Pro His Thr 35 40 45 Ser Arg Ile Ser Ser Val Ser Ser Gln PheSer Asp Gly Pro Ile Pro 50 55 60 Ser Pro Ser Ala Arg Ser Ser Ala Ser SerTrp Ser Glu Glu Pro Val 65 70 75 80 Gln Ser Asn Met Asp Ile Ser Thr GlyHis Met Ile Leu Ser Tyr Met 85 90 95 Glu Asp His Leu Lys Asn Lys Asn ArgLeu Glu Lys Glu Trp Glu Ala 100 105 110 Leu Cys Ala Tyr Gln Ala Glu ProAsn Ser Ser Phe Val Ala Gln Arg 115 120 125 Glu Glu Asn Val Pro Lys AsnArg Ser Leu Ala Val Leu Thr Tyr Asp 130 135 140 His Ser Arg Val Leu LeuLys Ala Glu Asn Ser His Ser His Ser Asp 145 150 155 160 Tyr Ile Asn AlaSer Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala 165 170 175 Tyr Ile AlaThr Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp 180 185 190 Gln MetVal Trp Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro 195 200 205 LeuAla Glu Asn Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu 210 215 220Gly Ser Asn Leu Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His 225 230235 240 Ile Trp Cys Glu Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu245 250 255 Gln Thr Asn Glu Thr Arg Thr Val Thr Gln Phe His Phe Leu SerTrp 260 265 270 Tyr Asp Arg Gly Val Pro Ser Ser Ser Arg Ser Leu Leu AspPhe Arg 275 280 285 Arg Lys Val Asn Lys Cys Tyr Arg Gly Arg Ser Cys ProIle Ile Val 290 295 300 His Cys Ser Asp Gly Ala Gly Arg Ser Gly Thr TyrVal Leu Ile Asp 305 310 315 320 Met Val Leu Asn Lys Met Ala Lys Gly AlaLys Glu Ile Asp Ile Ala 325 330 335 Ala Thr Leu Glu His Leu Arg Asp GlnArg Pro Gly Met Val Gln Thr 340 345 350 Lys Glu Gln Phe Glu Phe Ala LeuThr Ala Val Ala Glu Glu Val Asn 355 360 365 Ala Ile Leu Lys Ala Leu ProGln 370 375 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other(vii) IMMEDIATE SOURCE: (B) CLONE: ZC3747 (xi) SEQUENCE DESCRIPTION: SEQID NO: 8: GGGAATAACA TGTGAATGAC AAAATAAAAT GATAGCTTGC GCTTTTGCG 49 (2)INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other (vii) IMMEDIATE SOURCE: (B)CLONE: ZC8802 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GCGCCTCGAGCCACCATGCA GCATGCGCGG CAGCAAGAC 39 (2) INFORMATION FOR SEQ ID NO: 10:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: Other (vii) IMMEDIATE SOURCE: (B) CLONE: ZC8803 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 10: GCGCGAATTC TCACTGGGGC AGGGCCTTGA G 31 (2)INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: Other (vii) IMMEDIATE SOURCE: (B)CLONE: ZC10011 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TGTACGCGGGGTTCCTC 17 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other(vii) IMMEDIATE SOURCE: (B) CLONE: ZC10177 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 12: GAGGAACCCC GCGTACATCG CCACC 25 (2) INFORMATION FOR SEQ IDNO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 13: Val His Cys Xaa Ala Gly Xaa Xaa Arg Xaa Gly 1 5 10 (2)INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: AARGCNACNGTNGAYAAY 18 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 3287 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY: Coding Sequence (B) LOCATION: 4...3039 (D) OTHERINFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: AGG GCG CTC CCGCTG CTG TTG CTG CTA CTG CTG CTG CTG CCG CCA CGC 48 Ala Leu Pro Leu LeuLeu Leu Leu Leu Leu Leu Leu Pro Pro Arg 1 5 10 15 GTC CTG CCT GCC GCCCCC TCG TCC GTC CCC CAC GGC CGG CAG CTC CCG 96 Val Leu Pro Ala Ala ProSer Ser Val Pro His Gly Arg Gln Leu Pro 20 25 30 GGG CGC CTG GGC TGC CTACTC GAG GAG GGC CTC TGC GGA GCG TCC GAG 144 Gly Arg Leu Gly Cys Leu LeuGlu Glu Gly Leu Cys Gly Ala Ser Glu 35 40 45 GCC TGT GTG AAC GAT GGA GTGTTT GGA AGG TGC CAG AAG GTT CCG GCA 192 Ala Cys Val Asn Asp Gly Val PheGly Arg Cys Gln Lys Val Pro Ala 50 55 60 ATG GAC TTT TAC CGC TAC GAG GTGTCG CCC GTG GCC CTG CAG CGC CTG 240 Met Asp Phe Tyr Arg Tyr Glu Val SerPro Val Ala Leu Gln Arg Leu 65 70 75 CGC GTG GCT TTG CAG AAA CTC TCC GGCACA GGT TTC ACG TGG CAG GAT 288 Arg Val Ala Leu Gln Lys Leu Ser Gly ThrGly Phe Thr Trp Gln Asp 80 85 90 95 GAC TAT ACT CAG TAT GTG ATG GAC CAGGAA CTT GCA GAC CTC CCC AAA 336 Asp Tyr Thr Gln Tyr Val Met Asp Gln GluLeu Ala Asp Leu Pro Lys 100 105 110 ACC TAC CTG AGG CAT CCT GAA GCG TCCGGC CCA GCC AGG CCC TCA AAA 384 Thr Tyr Leu Arg His Pro Glu Ala Ser GlyPro Ala Arg Pro Ser Lys 115 120 125 CAC AGC ATT GGC AGT GAG AGG AGG TACAGT CGG GAG GGC GGC GCT GCC 432 His Ser Ile Gly Ser Glu Arg Arg Tyr SerArg Glu Gly Gly Ala Ala 130 135 140 CTG GCC AAG GCC TTC CGA CGC CAC CTGCCC TTC CTG GAG GCC CTG TCC 480 Leu Ala Lys Ala Phe Arg Arg His Leu ProPhe Leu Glu Ala Leu Ser 145 150 155 CAG GCC CCA GCT TCA GAC GCG CTC GCCAGG ACC CGG ATG GCG CAG GAC 528 Gln Ala Pro Ala Ser Asp Ala Leu Ala ArgThr Arg Met Ala Gln Asp 160 165 170 175 AGA CCC CGT GCT GAG GGT GAC GACCGC TTC TCC AAG AGC ATC CTG ACC 576 Arg Pro Arg Ala Glu Gly Asp Asp ArgPhe Ser Lys Ser Ile Leu Thr 180 185 190 TAT GTG GCC CAC ACG TCT GTG CTGACC TAC CCT CCC GGG CCC CAG GCC 624 Tyr Val Ala His Thr Ser Val Leu ThrTyr Pro Pro Gly Pro Gln Ala 195 200 205 CAG CTC CCC GAG GAC CTC CTG CCACGG ACC CTC AGC CAG CTC CAG CCA 672 Gln Leu Pro Glu Asp Leu Leu Pro ArgThr Leu Ser Gln Leu Gln Pro 210 215 220 GAC GAG CTC AGC CCT AAG GTG GACAGC AGT GTG GAG AGA CAC CAT CTG 720 Asp Glu Leu Ser Pro Lys Val Asp SerSer Val Glu Arg His His Leu 225 230 235 ATG GCA GCC CTC AGT GCC TAT GCTGCC CAG AGG CCC CCA GCT CCC CCT 768 Met Ala Ala Leu Ser Ala Tyr Ala AlaGln Arg Pro Pro Ala Pro Pro 240 245 250 255 GGG AAG GGC AGC CTG GAG CCGCAG TAC CTT CTG CGC GCC CCG TCC AGA 816 Gly Lys Gly Ser Leu Glu Pro GlnTyr Leu Leu Arg Ala Pro Ser Arg 260 265 270 ATG CCC AGG CCC TTG TTG TCGCCA GCC GTC CCC CAG AAG TGG CCT TCA 864 Met Pro Arg Pro Leu Leu Ser ProAla Val Pro Gln Lys Trp Pro Ser 275 280 285 CCT CTG GGA GAT CCT GAA GACCCC CCC AGC ACA GGG GAA GGA GCA CGG 912 Pro Leu Gly Asp Pro Glu Asp ProPro Ser Thr Gly Glu Gly Ala Arg 290 295 300 ATT CAC ACT CTC CTG AAG GACCTG CAG AGG CAG CCG GCT GAG GCG AGG 960 Ile His Thr Leu Leu Lys Asp LeuGln Arg Gln Pro Ala Glu Ala Arg 305 310 315 GGC CTG AGT GAC CTG GAG CTGGAC AGC ATG GCC GAG CTG ATG GCT GGC 1008 Gly Leu Ser Asp Leu Glu Leu AspSer Met Ala Glu Leu Met Ala Gly 320 325 330 335 CTG ATG CAA GGC ATG GACCAC AGA GGA GCT CTA GGC GGC CCT GGG AAA 1056 Leu Met Gln Gly Met Asp HisArg Gly Ala Leu Gly Gly Pro Gly Lys 340 345 350 GCG GCC CTG GGA GAG TCTGGA GAA CAG GCG GAT GGC CCC AAG GCC GCC 1104 Ala Ala Leu Gly Glu Ser GlyGlu Gln Ala Asp Gly Pro Lys Ala Ala 355 360 365 CTC CGT GGG GAA AGC TTTCCA GAT GAC GGA GTT CAG GAC GAC GAT GAC 1152 Leu Arg Gly Glu Ser Phe ProAsp Asp Gly Val Gln Asp Asp Asp Asp 370 375 380 AGA CTT TAC CAA GAG GTCCAT CGT CTG AGT GCC ACA CTC GGG GGC CTC 1200 Arg Leu Tyr Gln Glu Val HisArg Leu Ser Ala Thr Leu Gly Gly Leu 385 390 395 CTG CAG GAC CAC GGG TCTCGA CTC TCG CCT GGA GCC CTC CCC TTT GCA 1248 Leu Gln Asp His Gly Ser ArgLeu Ser Pro Gly Ala Leu Pro Phe Ala 400 405 410 415 AAG CCC CTC AAA ATGGAG AGG AAG AAA TCC GAG CGC CCT GAG GCT TCC 1296 Lys Pro Leu Lys Met GluArg Lys Lys Ser Glu Arg Pro Glu Ala Ser 420 425 430 CTG TCT TCA GAA GAGGAG ACT GCC GGA GTG GAG AAC GTC AAG AGC CAG 1344 Leu Ser Ser Glu Glu GluThr Ala Gly Val Glu Asn Val Lys Ser Gln 435 440 445 ACG TAT TCC AAA GACCTG CTG GGG CAG CAG CCG CAT TCG GAG CCC GGG 1392 Thr Tyr Ser Lys Asp LeuLeu Gly Gln Gln Pro His Ser Glu Pro Gly 450 455 460 GCA GGC GCG TTT GGGGAG CTC CAA AAC CAG ATG CCT GGG CCC TCG GAG 1440 Ala Gly Ala Phe Gly GluLeu Gln Asn Gln Met Pro Gly Pro Ser Glu 465 470 475 GAG GAG CAG AGC CTTCCA GCG GGT GCT CAG GAG GCC CTC GGC GAC GGC 1488 Glu Glu Gln Ser Leu ProAla Gly Ala Gln Glu Ala Leu Gly Asp Gly 480 485 490 495 CTG CAA TTG GAAGTC AAG CCT TCC GAG GAA GAG GCA CGG TGC TAC ATC 1536 Leu Gln Leu Glu ValLys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile 500 505 510 GTG ACA GAC AGAGAC CCC CTG CGC CCC GAG GAA GGA AGG CAG CTG GTG 1584 Val Thr Asp Arg AspPro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val 515 520 525 GAG GAC GTC GCCCGC CTC CTG CAG ATG CCC AGC AGC ACA TTC GCC GAC 1632 Glu Asp Val Ala ArgLeu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp 530 535 540 GTG GAG GTT CTCGGA CCA GCA GTG ACC TTC AAA GTG GGC GCC AAT GTC 1680 Val Glu Val Leu GlyPro Ala Val Thr Phe Lys Val Gly Ala Asn Val 545 550 555 CAG AAC GTG ACCACT GCG GAT GTG GAG AAG GCC ACA GTT GAC AAC AAA 1728 Gln Asn Val Thr ThrAla Asp Val Glu Lys Ala Thr Val Asp Asn Lys 560 565 570 575 GAC AAA CTGGAG GAA ACC TCT GGA CTG AAA ATT CTT CAA ACC GGA GTC 1776 Asp Lys Leu GluGlu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val 580 585 590 GGG TCG AAAAGC AAA CTC AAG TTC CTG CCT CCT CAG GCG GAG CAA GAA 1824 Gly Ser Lys SerLys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu 595 600 605 GAC TCA ACCAAG TTC ATC GCG CTC ACC CTG GTC TCC CTC GCC TGC ATC 1872 Asp Ser Thr LysPhe Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile 610 615 620 CTG GGC GTCCTC CTG GCC TCT GGC CTC ATC TAC TGC CTA CGC CAT AGC 1920 Leu Gly Val LeuLeu Ala Ser Gly Leu Ile Tyr Cys Leu Arg His Ser 625 630 635 TCT CAG CACAGG CTG AAG GAG AAG CTC TCG GGA CTA GGG CGC GAC CCA 1968 Ser Gln His ArgLeu Lys Glu Lys Leu Ser Gly Leu Gly Arg Asp Pro 640 645 650 655 GGT GCAGAT GCC ACC GCC GCC TAC CAG GAG CTG TGC CGC CAG CGT ATG 2016 Gly Ala AspAla Thr Ala Ala Tyr Gln Glu Leu Cys Arg Gln Arg Met 660 665 670 GCC ACGCGG CCA CCA GAC CGG CCC GAG GGC CCG CAC ACA TCC CGC ATC 2064 Ala Thr ArgPro Pro Asp Arg Pro Glu Gly Pro His Thr Ser Arg Ile 675 680 685 AGC AGCGTC TCG TCC CAG TTC AGC GAC GGG CCG ATG CCC AGC CCC TCC 2112 Ser Ser ValSer Ser Gln Phe Ser Asp Gly Pro Met Pro Ser Pro Ser 690 695 700 GCA CGCAGC AGC GCC TCG TCC TGG TCC GAG GAG CCC GTG CAG TCC AAC 2160 Ala Arg SerSer Ala Ser Ser Trp Ser Glu Glu Pro Val Gln Ser Asn 705 710 715 ATG GACATC TCC ACC GGC CAC ATG ATC CTG TCC TAC ATG GAG GAC CAC 2208 Met Asp IleSer Thr Gly His Met Ile Leu Ser Tyr Met Glu Asp His 720 725 730 735 CTGAAG AAC AAG AAC CGG CTG GAG AAG GAG TGG GAG GCG CTG TGT GCC 2256 Leu LysAsn Lys Asn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala 740 745 750 TACCAG GCG GAG CCC AAC AGC TCA CTT GTG GCC CAG AAG GAG GAG AAT 2304 Tyr GlnAla Glu Pro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn 755 760 765 GTGCCC AAG AAC CGC TCC CTG GCC GTG CTG ACC TAT GAC CAC TCC CGG 2352 Val ProLys Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg 770 775 780 GTCCTA CTG AAG GCG GAG AAC AGC CAC AGC CAC TCG GAC TAC ATC AAC 2400 Val LeuLeu Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn 785 790 795 GCCAGC CCC ATC ATG GAT CAC GAC CCG AGG AAC CCC GCG TAC ATC GCC 2448 Ala SerPro Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala 800 805 810 815ACC CAG GGA CCG CTG CCC GCC ACC GTG GCC GAC TTT TGG CAG ATG GTG 2496 ThrGln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val 820 825 830TGG GAG AGC GGC TGC GTG GTG ATC GTC ATG CTG ACA CCC CTC ACA GAG 2544 TrpGlu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Thr Glu 835 840 845AAC GGC GTC CGG CAG TGC TAC CAC TAC TGG CCA GAT GAA GGC TCC AAC 2592 AsnGly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn 850 855 860CTC TAC CAC ATC TAT GAG GTG AAC CTG GTC TCC GAG CAC ATC TGG TGC 2640 LeuTyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp Cys 865 870 875GAG GAC TTT CTG GTG AGG AGC TTC TAT CTG AAG AAC CTG CAG ACC AAC 2688 GluAsp Phe Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu Gln Thr Asn 880 885 890895 GAG ACG CGC ACC GTG ACC CAG TTC CAC TTC CTG AGT TGG TAT GAC CGA 2736Glu Thr Arg Thr Val Thr Gln Phe His Phe Leu Ser Trp Tyr Asp Arg 900 905910 GGA GTC CCC TCC TCC TCA AGA TCC CTC CTG GAC TTC CGC AGA AAA GTA 2784Gly Val Pro Ser Ser Ser Arg Ser Leu Leu Asp Phe Arg Arg Lys Val 915 920925 AAC AAG TGC TAC AGG GGC CGT TCT TGT CCA ATA ATT GTT CAT TGC AGT 2832Asn Lys Cys Tyr Arg Gly Arg Ser Cys Pro Ile Ile Val His Cys Ser 930 935940 GAC GGT GCA GGC CGG AGC GGC ACC TAC GTC CTG ATC GAC ATG GTT CTC 2880Asp Gly Ala Gly Arg Ser Gly Thr Tyr Val Leu Ile Asp Met Val Leu 945 950955 AAC AAG ATG GCC AAA GGT GCT AAA GAG ATT GAT ATC GCA GCA ACC CTG 2928Asn Lys Met Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu 960 965970 975 GAG CAC TTG AGG GAC CAG AGA CCC GGC ATG GTC CAG ACG AAG GAG CAG2976 Glu His Leu Arg Asp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln 980985 990 TTT GAG TTC GCG CTG ACA GCC GTG GCT GAA GAG GTG AAT GCC ATC CTC3024 Phe Glu Phe Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu 9951000 1005 AAG GCC CTT CCC CAG TGAGCAGCGG CCTCGGGGCC TCGGGGGAGCCCCCACCCCC C 3080 Lys Ala Leu Pro Gln 1010 GGATGTCGTC AGGAATCGTGATCTGACTTT AATTGTGTGT CTTCTATTAT AACTGCATAG 3140 TAATAGGGCC CTTAGCTCTCCCGTAGTCAG CGCAGTTTAG CAGTTAAGCA GTTAAAATGT 3200 GTATTTTTGT TTAATCCAACAATAATAAAG AGAGATTTGT GGAAAAATCC CAAAAAAAAA 3260 AAAAAAAAAA AAAAAAAAAAACTCGAG 3287 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1012 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:Ala Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Pro Pro Arg Val 1 5 1015 Leu Pro Ala Ala Pro Ser Ser Val Pro His Gly Arg Gln Leu Pro Gly 20 2530 Arg Leu Gly Cys Leu Leu Glu Glu Gly Leu Cys Gly Ala Ser Glu Ala 35 4045 Cys Val Asn Asp Gly Val Phe Gly Arg Cys Gln Lys Val Pro Ala Met 50 5560 Asp Phe Tyr Arg Tyr Glu Val Ser Pro Val Ala Leu Gln Arg Leu Arg 65 7075 80 Val Ala Leu Gln Lys Leu Ser Gly Thr Gly Phe Thr Trp Gln Asp Asp 8590 95 Tyr Thr Gln Tyr Val Met Asp Gln Glu Leu Ala Asp Leu Pro Lys Thr100 105 110 Tyr Leu Arg His Pro Glu Ala Ser Gly Pro Ala Arg Pro Ser LysHis 115 120 125 Ser Ile Gly Ser Glu Arg Arg Tyr Ser Arg Glu Gly Gly AlaAla Leu 130 135 140 Ala Lys Ala Phe Arg Arg His Leu Pro Phe Leu Glu AlaLeu Ser Gln 145 150 155 160 Ala Pro Ala Ser Asp Ala Leu Ala Arg Thr ArgMet Ala Gln Asp Arg 165 170 175 Pro Arg Ala Glu Gly Asp Asp Arg Phe SerLys Ser Ile Leu Thr Tyr 180 185 190 Val Ala His Thr Ser Val Leu Thr TyrPro Pro Gly Pro Gln Ala Gln 195 200 205 Leu Pro Glu Asp Leu Leu Pro ArgThr Leu Ser Gln Leu Gln Pro Asp 210 215 220 Glu Leu Ser Pro Lys Val AspSer Ser Val Glu Arg His His Leu Met 225 230 235 240 Ala Ala Leu Ser AlaTyr Ala Ala Gln Arg Pro Pro Ala Pro Pro Gly 245 250 255 Lys Gly Ser LeuGlu Pro Gln Tyr Leu Leu Arg Ala Pro Ser Arg Met 260 265 270 Pro Arg ProLeu Leu Ser Pro Ala Val Pro Gln Lys Trp Pro Ser Pro 275 280 285 Leu GlyAsp Pro Glu Asp Pro Pro Ser Thr Gly Glu Gly Ala Arg Ile 290 295 300 HisThr Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu Ala Arg Gly 305 310 315320 Leu Ser Asp Leu Glu Leu Asp Ser Met Ala Glu Leu Met Ala Gly Leu 325330 335 Met Gln Gly Met Asp His Arg Gly Ala Leu Gly Gly Pro Gly Lys Ala340 345 350 Ala Leu Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro Lys Ala AlaLeu 355 360 365 Arg Gly Glu Ser Phe Pro Asp Asp Gly Val Gln Asp Asp AspAsp Arg 370 375 380 Leu Tyr Gln Glu Val His Arg Leu Ser Ala Thr Leu GlyGly Leu Leu 385 390 395 400 Gln Asp His Gly Ser Arg Leu Ser Pro Gly AlaLeu Pro Phe Ala Lys 405 410 415 Pro Leu Lys Met Glu Arg Lys Lys Ser GluArg Pro Glu Ala Ser Leu 420 425 430 Ser Ser Glu Glu Glu Thr Ala Gly ValGlu Asn Val Lys Ser Gln Thr 435 440 445 Tyr Ser Lys Asp Leu Leu Gly GlnGln Pro His Ser Glu Pro Gly Ala 450 455 460 Gly Ala Phe Gly Glu Leu GlnAsn Gln Met Pro Gly Pro Ser Glu Glu 465 470 475 480 Glu Gln Ser Leu ProAla Gly Ala Gln Glu Ala Leu Gly Asp Gly Leu 485 490 495 Gln Leu Glu ValLys Pro Ser Glu Glu Glu Ala Arg Cys Tyr Ile Val 500 505 510 Thr Asp ArgAsp Pro Leu Arg Pro Glu Glu Gly Arg Gln Leu Val Glu 515 520 525 Asp ValAla Arg Leu Leu Gln Met Pro Ser Ser Thr Phe Ala Asp Val 530 535 540 GluVal Leu Gly Pro Ala Val Thr Phe Lys Val Gly Ala Asn Val Gln 545 550 555560 Asn Val Thr Thr Ala Asp Val Glu Lys Ala Thr Val Asp Asn Lys Asp 565570 575 Lys Leu Glu Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly Val Gly580 585 590 Ser Lys Ser Lys Leu Lys Phe Leu Pro Pro Gln Ala Glu Gln GluAsp 595 600 605 Ser Thr Lys Phe Ile Ala Leu Thr Leu Val Ser Leu Ala CysIle Leu 610 615 620 Gly Val Leu Leu Ala Ser Gly Leu Ile Tyr Cys Leu ArgHis Ser Ser 625 630 635 640 Gln His Arg Leu Lys Glu Lys Leu Ser Gly LeuGly Arg Asp Pro Gly 645 650 655 Ala Asp Ala Thr Ala Ala Tyr Gln Glu LeuCys Arg Gln Arg Met Ala 660 665 670 Thr Arg Pro Pro Asp Arg Pro Glu GlyPro His Thr Ser Arg Ile Ser 675 680 685 Ser Val Ser Ser Gln Phe Ser AspGly Pro Met Pro Ser Pro Ser Ala 690 695 700 Arg Ser Ser Ala Ser Ser TrpSer Glu Glu Pro Val Gln Ser Asn Met 705 710 715 720 Asp Ile Ser Thr GlyHis Met Ile Leu Ser Tyr Met Glu Asp His Leu 725 730 735 Lys Asn Lys AsnArg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr 740 745 750 Gln Ala GluPro Asn Ser Ser Leu Val Ala Gln Lys Glu Glu Asn Val 755 760 765 Pro LysAsn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val 770 775 780 LeuLeu Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala 785 790 795800 Ser Pro Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala Thr 805810 815 Gln Gly Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met Val Trp820 825 830 Glu Ser Gly Cys Val Val Ile Val Met Leu Thr Pro Leu Thr GluAsn 835 840 845 Gly Val Arg Gln Cys Tyr His Tyr Trp Pro Asp Glu Gly SerAsn Leu 850 855 860 Tyr His Ile Tyr Glu Val Asn Leu Val Ser Glu His IleTrp Cys Glu 865 870 875 880 Asp Phe Leu Val Arg Ser Phe Tyr Leu Lys AsnLeu Gln Thr Asn Glu 885 890 895 Thr Arg Thr Val Thr Gln Phe His Phe LeuSer Trp Tyr Asp Arg Gly 900 905 910 Val Pro Ser Ser Ser Arg Ser Leu LeuAsp Phe Arg Arg Lys Val Asn 915 920 925 Lys Cys Tyr Arg Gly Arg Ser CysPro Ile Ile Val His Cys Ser Asp 930 935 940 Gly Ala Gly Arg Ser Gly ThrTyr Val Leu Ile Asp Met Val Leu Asn 945 950 955 960 Lys Met Ala Lys GlyAla Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu 965 970 975 His Leu Arg AspGln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe 980 985 990 Glu Phe AlaLeu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys 995 1000 1005 AlaLeu Pro Gln 1010 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Other(vii) IMMEDIATE SOURCE: (B) CLONE: ZC11653 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 17: CGGAATTCCT CTGTGGTCCA TGCCTTGC 28 (2) INFORMATION FOR SEQID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GCGCCATGAACTTGGTGGAG TCTTCTTGCT CCGCCTGA 38 (2) INFORMATION FOR SEQ ID NO: 19: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AGCTGCCTCC TCCCTCTGTCCCACTCCTGT CTGCAAGA 38 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 20: CTTGGGTGTG TAGAAGAAGC CACGTTCCCC 30(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 2464 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: Coding Sequence (B) LOCATION: 2...2455 (D) OTHER INFORMATION:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: T CAC ACG TCT GTG CTG ACC TACCCT CCC GGG CCC CGG ACC CAG CTC CAC 49 His Thr Ser Val Leu Thr Tyr ProPro Gly Pro Arg Thr Gln Leu His 1 5 10 15 GAG GAC CTC CTG CCA CGG ACCCTC GGC CAG CTC CAG CCA GAT GAG CTC 97 Glu Asp Leu Leu Pro Arg Thr LeuGly Gln Leu Gln Pro Asp Glu Leu 20 25 30 AGC CCT AAG GTG GAC AGT GGT GTGGAC AGA CAC CAT CTG ATG GCG GCC 145 Ser Pro Lys Val Asp Ser Gly Val AspArg His His Leu Met Ala Ala 35 40 45 CTC AGT GCC TAT GCT GCC CAG AGG CCCCCA GCT CCC CCC GGG GAG GGC 193 Leu Ser Ala Tyr Ala Ala Gln Arg Pro ProAla Pro Pro Gly Glu Gly 50 55 60 AGC CTG GAG CCA CAG TAC CTT CTG CGT GCACCC TCA AGA ATG CCC AGG 241 Ser Leu Glu Pro Gln Tyr Leu Leu Arg Ala ProSer Arg Met Pro Arg 65 70 75 80 CCT TTG CTG GCA CCA GCC GCC CCC CAG AAGTGG CCT TCA CCT CTG GGA 289 Pro Leu Leu Ala Pro Ala Ala Pro Gln Lys TrpPro Ser Pro Leu Gly 85 90 95 GAT TCC GAA GAC CCC TCT AGC ACA GGC GAT GGAGCA CGG ATT CAT ACC 337 Asp Ser Glu Asp Pro Ser Ser Thr Gly Asp Gly AlaArg Ile His Thr 100 105 110 CTC CTG AAG GAC CTG CAG AGG CAG CCG GCT GAGGTG AGG GGC CTG AGT 385 Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu ValArg Gly Leu Ser 115 120 125 GGC CTG GAG CTG GAC GGC ATG GCT GAG CTG ATGGCT GGC CTG ATG CAA 433 Gly Leu Glu Leu Asp Gly Met Ala Glu Leu Met AlaGly Leu Met Gln 130 135 140 GGC GTG GAC CAT GGA GTA GCT CGA GGC AGC CCTGGG AGA GCG GCC CTG 481 Gly Val Asp His Gly Val Ala Arg Gly Ser Pro GlyArg Ala Ala Leu 145 150 155 160 GGA GAG TCT GGA GAA CAG GCG GAT GGC CCCAAG GCC ACC CTC CGT GGA 529 Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro LysAla Thr Leu Arg Gly 165 170 175 GAC AGC TTT CCA GAT GAC GGA GTG CAG GACGAC GAT GAT AGA CTT TAC 577 Asp Ser Phe Pro Asp Asp Gly Val Gln Asp AspAsp Asp Arg Leu Tyr 180 185 190 CAA GAG GTC CAT CGT CTG AGT GCC ACA CTCGGG GGC CTC CTG CAG GAC 625 Gln Glu Val His Arg Leu Ser Ala Thr Leu GlyGly Leu Leu Gln Asp 195 200 205 CAC GGG TCT CGA CTC TTA CCT GGA GCC CTCCCC TTT GCA AGG CCC CTC 673 His Gly Ser Arg Leu Leu Pro Gly Ala Leu ProPhe Ala Arg Pro Leu 210 215 220 GAC ATG GAG AGG AAG AAG TCC GAG CAC CCTGAG TCT TCC CTG TCT TCA 721 Asp Met Glu Arg Lys Lys Ser Glu His Pro GluSer Ser Leu Ser Ser 225 230 235 240 GAA GAG GAG ACT GCC GGA GTG GAG AACGTC AAG AGC CAG ACG TAT TCC 769 Glu Glu Glu Thr Ala Gly Val Glu Asn ValLys Ser Gln Thr Tyr Ser 245 250 255 AAA GAT CTG CTG GGG CGG CAG CCG CATTCG GAG CCC GGG GCC GCT GCG 817 Lys Asp Leu Leu Gly Arg Gln Pro His SerGlu Pro Gly Ala Ala Ala 260 265 270 TTT GGG GAG CTC CAA AAC CAG ATG CCTGGG CCC TCG AAG GAG GAG CAG 865 Phe Gly Glu Leu Gln Asn Gln Met Pro GlyPro Ser Lys Glu Glu Gln 275 280 285 AGC CTT CCA GCG GGT GCT CAG GAG GCCCTC AGC GAC GGC CTG CAA TTG 913 Ser Leu Pro Ala Gly Ala Gln Glu Ala LeuSer Asp Gly Leu Gln Leu 290 295 300 GAG GTC CAG CCT TCC GAG GAA GAG GCGCGG GGC TAC ATC GTG ACA GAC 961 Glu Val Gln Pro Ser Glu Glu Glu Ala ArgGly Tyr Ile Val Thr Asp 305 310 315 320 GGA GAC CCC CTG CGC CCC GAG GAAGGA AGG CGG CTG GTG GAG GAC GTC 1009 Gly Asp Pro Leu Arg Pro Glu Glu GlyArg Arg Leu Val Glu Asp Val 325 330 335 GCC CGC CTC CTG CAG GTG CCC AGCAGC GCG TTC GCT GAC GTG GAG GTT 1057 Ala Arg Leu Leu Gln Val Pro Ser SerAla Phe Ala Asp Val Glu Val 340 345 350 CTC GGA CCA GCA GTG ACC TTC AAAGTG AGC GCC AAT GTC CAA AAC GTG 1105 Leu Gly Pro Ala Val Thr Phe Lys ValSer Ala Asn Val Gln Asn Val 355 360 365 ACC ACT GAG GAT GTG GAG AAG GCCACA GTT GAC AAC AAA GAC AAA CTG 1153 Thr Thr Glu Asp Val Glu Lys Ala ThrVal Asp Asn Lys Asp Lys Leu 370 375 380 GAG GAA ACC TCT GGA CTG AAA ATTCTT CAA ACC GGA GTC GGG TCG AAA 1201 Glu Glu Thr Ser Gly Leu Lys Ile LeuGln Thr Gly Val Gly Ser Lys 385 390 395 400 AGC AAA CTC AAG TTC CTG CCTCCT CAG GCG GAG CAA GAA GAC TCC ACC 1249 Ser Lys Leu Lys Phe Leu Pro ProGln Ala Glu Gln Glu Asp Ser Thr 405 410 415 AAG TTC ATC GCG CTC ACC CTGGTC TCC CTC GCC TGC ATC CTG GGC GTC 1297 Lys Phe Ile Ala Leu Thr Leu ValSer Leu Ala Cys Ile Leu Gly Val 420 425 430 CTC CTG GCC TCT GGC CTC ATCTAC TGC CTC CGC CAT AGC TCT CAG CAC 1345 Leu Leu Ala Ser Gly Leu Ile TyrCys Leu Arg His Ser Ser Gln His 435 440 445 AGG CTG AAG GAG AAG CTC TCGGGA CTA GGG GGC GAC CCA GGT GCA GAT 1393 Arg Leu Lys Glu Lys Leu Ser GlyLeu Gly Gly Asp Pro Gly Ala Asp 450 455 460 GCC ACT GCC GCC TAC CAG GAGCTG TGC CGC CAG CGT ATG GCC ACG CGG 1441 Ala Thr Ala Ala Tyr Gln Glu LeuCys Arg Gln Arg Met Ala Thr Arg 465 470 475 480 CCA CCA GAC CGA CCT GAGGGC CCG CAC ACG TCA CGC ATC AGC AGC GTC 1489 Pro Pro Asp Arg Pro Glu GlyPro His Thr Ser Arg Ile Ser Ser Val 485 490 495 TCA TCC CAG TTC AGC GACGGG CCG ATC CCC AGC CCC TCC GCA CGC AGC 1537 Ser Ser Gln Phe Ser Asp GlyPro Ile Pro Ser Pro Ser Ala Arg Ser 500 505 510 AGC GCC TCA TCC TGG TCCGAG GAG CCT GTG CAG TCC AAC ATG GAC ATC 1585 Ser Ala Ser Ser Trp Ser GluGlu Pro Val Gln Ser Asn Met Asp Ile 515 520 525 TCC ACC GGC CAC ATG ATCCTG TCC TAC ATG GAG GAC CAC CTG AAG AAC 1633 Ser Thr Gly His Met Ile LeuSer Tyr Met Glu Asp His Leu Lys Asn 530 535 540 AAG AAC CGG CTG GAG AAGGAG TGG GAA GCG CTG TGC GCC TAC CAG GCG 1681 Lys Asn Arg Leu Glu Lys GluTrp Glu Ala Leu Cys Ala Tyr Gln Ala 545 550 555 560 GAG CCC AAC AGC TCGTTC GTG GCC CAG AGG GAG GAG AAC GTG CCC AAG 1729 Glu Pro Asn Ser Ser PheVal Ala Gln Arg Glu Glu Asn Val Pro Lys 565 570 575 AAC CGC TCC CTG GCCGTG CTG ACC TAT GAC CAC TCC CGG GTC CTG CTG 1777 Asn Arg Ser Leu Ala ValLeu Thr Tyr Asp His Ser Arg Val Leu Leu 580 585 590 AAG GCG GAG AAC AGCCAC AGC CAC TCA GAC TAC ATC AAC GCT AGC CCC 1825 Lys Ala Glu Asn Ser HisSer His Ser Asp Tyr Ile Asn Ala Ser Pro 595 600 605 ATC ATG GAT CAC GACCCG AGG AAC CCC GCG TAC ATC GCC ACC CAG GGA 1873 Ile Met Asp His Asp ProArg Asn Pro Ala Tyr Ile Ala Thr Gln Gly 610 615 620 CCG CTG CCC GCC ACCGTG GCT GAC TTT TGG CAG ATG GTG TGG GAG AGC 1921 Pro Leu Pro Ala Thr ValAla Asp Phe Trp Gln Met Val Trp Glu Ser 625 630 635 640 GGC TGC GTG GTGATC GTC ATG CTG ACA CCC CTC GCG GAG AAC GGC GTC 1969 Gly Cys Val Val IleVal Met Leu Thr Pro Leu Ala Glu Asn Gly Val 645 650 655 CGG CAG TGC TACCAC TAC TGG CCG GAT GAA GGC TCC AAT CTC TAC CAC 2017 Arg Gln Cys Tyr HisTyr Trp Pro Asp Glu Gly Ser Asn Leu Tyr His 660 665 670 ATC TAT GAG GTGAAC CTG GTC TCC GAG CAC ATC TGG TGT GAG GAC TTC 2065 Ile Tyr Glu Val AsnLeu Val Ser Glu His Ile Trp Cys Glu Asp Phe 675 680 685 CTG GTG AGG AGCTTC TAT CTG AAG AAC CTG CAG ACC AAC GAG ACG CGC 2113 Leu Val Arg Ser PheTyr Leu Lys Asn Leu Gln Thr Asn Glu Thr Arg 690 695 700 ACC GTG ACG CAGTTC CAC TTC CTG AGT TGG TAT GAC CGA GGA GTC CCT 2161 Thr Val Thr Gln PheHis Phe Leu Ser Trp Tyr Asp Arg Gly Val Pro 705 710 715 720 TCC TCC TCAAGG TCC CTC CTG GAC TTC CGC AGA AAA GTA AAC AAG TGC 2209 Ser Ser Ser ArgSer Leu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys 725 730 735 TAC AGG GGCCGT TCT TGT CCA ATA ATT GTT CAT TGC AGT GAC GGT GCA 2257 Tyr Arg Gly ArgSer Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala 740 745 750 GGC CGG AGCGGC ACC TAC GTC CTG ATC GAC ATG GTT CTC AAC AAG ATG 2305 Gly Arg Ser GlyThr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met 755 760 765 GCC AAA GGTGCT AAA GAG ATT GAT ATC GCA GCG ACC CTG GAG CAC TTG 2353 Ala Lys Gly AlaLys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu 770 775 780 AGG GAC CAGAGA CCC GGC ATG GTC CAG ACG AAG GAG CAG TTT GAG TTC 2401 Arg Asp Gln ArgPro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe 785 790 795 800 GCG CTGACA GCC GTG GCT GAG GAG GTG AAC GCC ATC CTC AAG GCC CTG 2449 Ala Leu ThrAla Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu 805 810 815 CCC CAGTGAGAATTC 2464 Pro Gln (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 818 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:His Thr Ser Val Leu Thr Tyr Pro Pro Gly Pro Arg Thr Gln Leu His 1 5 1015 Glu Asp Leu Leu Pro Arg Thr Leu Gly Gln Leu Gln Pro Asp Glu Leu 20 2530 Ser Pro Lys Val Asp Ser Gly Val Asp Arg His His Leu Met Ala Ala 35 4045 Leu Ser Ala Tyr Ala Ala Gln Arg Pro Pro Ala Pro Pro Gly Glu Gly 50 5560 Ser Leu Glu Pro Gln Tyr Leu Leu Arg Ala Pro Ser Arg Met Pro Arg 65 7075 80 Pro Leu Leu Ala Pro Ala Ala Pro Gln Lys Trp Pro Ser Pro Leu Gly 8590 95 Asp Ser Glu Asp Pro Ser Ser Thr Gly Asp Gly Ala Arg Ile His Thr100 105 110 Leu Leu Lys Asp Leu Gln Arg Gln Pro Ala Glu Val Arg Gly LeuSer 115 120 125 Gly Leu Glu Leu Asp Gly Met Ala Glu Leu Met Ala Gly LeuMet Gln 130 135 140 Gly Val Asp His Gly Val Ala Arg Gly Ser Pro Gly ArgAla Ala Leu 145 150 155 160 Gly Glu Ser Gly Glu Gln Ala Asp Gly Pro LysAla Thr Leu Arg Gly 165 170 175 Asp Ser Phe Pro Asp Asp Gly Val Gln AspAsp Asp Asp Arg Leu Tyr 180 185 190 Gln Glu Val His Arg Leu Ser Ala ThrLeu Gly Gly Leu Leu Gln Asp 195 200 205 His Gly Ser Arg Leu Leu Pro GlyAla Leu Pro Phe Ala Arg Pro Leu 210 215 220 Asp Met Glu Arg Lys Lys SerGlu His Pro Glu Ser Ser Leu Ser Ser 225 230 235 240 Glu Glu Glu Thr AlaGly Val Glu Asn Val Lys Ser Gln Thr Tyr Ser 245 250 255 Lys Asp Leu LeuGly Arg Gln Pro His Ser Glu Pro Gly Ala Ala Ala 260 265 270 Phe Gly GluLeu Gln Asn Gln Met Pro Gly Pro Ser Lys Glu Glu Gln 275 280 285 Ser LeuPro Ala Gly Ala Gln Glu Ala Leu Ser Asp Gly Leu Gln Leu 290 295 300 GluVal Gln Pro Ser Glu Glu Glu Ala Arg Gly Tyr Ile Val Thr Asp 305 310 315320 Gly Asp Pro Leu Arg Pro Glu Glu Gly Arg Arg Leu Val Glu Asp Val 325330 335 Ala Arg Leu Leu Gln Val Pro Ser Ser Ala Phe Ala Asp Val Glu Val340 345 350 Leu Gly Pro Ala Val Thr Phe Lys Val Ser Ala Asn Val Gln AsnVal 355 360 365 Thr Thr Glu Asp Val Glu Lys Ala Thr Val Asp Asn Lys AspLys Leu 370 375 380 Glu Glu Thr Ser Gly Leu Lys Ile Leu Gln Thr Gly ValGly Ser Lys 385 390 395 400 Ser Lys Leu Lys Phe Leu Pro Pro Gln Ala GluGln Glu Asp Ser Thr 405 410 415 Lys Phe Ile Ala Leu Thr Leu Val Ser LeuAla Cys Ile Leu Gly Val 420 425 430 Leu Leu Ala Ser Gly Leu Ile Tyr CysLeu Arg His Ser Ser Gln His 435 440 445 Arg Leu Lys Glu Lys Leu Ser GlyLeu Gly Gly Asp Pro Gly Ala Asp 450 455 460 Ala Thr Ala Ala Tyr Gln GluLeu Cys Arg Gln Arg Met Ala Thr Arg 465 470 475 480 Pro Pro Asp Arg ProGlu Gly Pro His Thr Ser Arg Ile Ser Ser Val 485 490 495 Ser Ser Gln PheSer Asp Gly Pro Ile Pro Ser Pro Ser Ala Arg Ser 500 505 510 Ser Ala SerSer Trp Ser Glu Glu Pro Val Gln Ser Asn Met Asp Ile 515 520 525 Ser ThrGly His Met Ile Leu Ser Tyr Met Glu Asp His Leu Lys Asn 530 535 540 LysAsn Arg Leu Glu Lys Glu Trp Glu Ala Leu Cys Ala Tyr Gln Ala 545 550 555560 Glu Pro Asn Ser Ser Phe Val Ala Gln Arg Glu Glu Asn Val Pro Lys 565570 575 Asn Arg Ser Leu Ala Val Leu Thr Tyr Asp His Ser Arg Val Leu Leu580 585 590 Lys Ala Glu Asn Ser His Ser His Ser Asp Tyr Ile Asn Ala SerPro 595 600 605 Ile Met Asp His Asp Pro Arg Asn Pro Ala Tyr Ile Ala ThrGln Gly 610 615 620 Pro Leu Pro Ala Thr Val Ala Asp Phe Trp Gln Met ValTrp Glu Ser 625 630 635 640 Gly Cys Val Val Ile Val Met Leu Thr Pro LeuAla Glu Asn Gly Val 645 650 655 Arg Gln Cys Tyr His Tyr Trp Pro Asp GluGly Ser Asn Leu Tyr His 660 665 670 Ile Tyr Glu Val Asn Leu Val Ser GluHis Ile Trp Cys Glu Asp Phe 675 680 685 Leu Val Arg Ser Phe Tyr Leu LysAsn Leu Gln Thr Asn Glu Thr Arg 690 695 700 Thr Val Thr Gln Phe His PheLeu Ser Trp Tyr Asp Arg Gly Val Pro 705 710 715 720 Ser Ser Ser Arg SerLeu Leu Asp Phe Arg Arg Lys Val Asn Lys Cys 725 730 735 Tyr Arg Gly ArgSer Cys Pro Ile Ile Val His Cys Ser Asp Gly Ala 740 745 750 Gly Arg SerGly Thr Tyr Val Leu Ile Asp Met Val Leu Asn Lys Met 755 760 765 Ala LysGly Ala Lys Glu Ile Asp Ile Ala Ala Thr Leu Glu His Leu 770 775 780 ArgAsp Gln Arg Pro Gly Met Val Gln Thr Lys Glu Gln Phe Glu Phe 785 790 795800 Ala Leu Thr Ala Val Ala Glu Glu Val Asn Ala Ile Leu Lys Ala Leu 805810 815 Pro Gln (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 2736 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 23: CAGGACAGAC CCCGTGCTGA GGGTGACGACCGCTTCTCCA AGAGCATCCT GACCTATGTG 60 GCCCACACGT CTGTGCTGAC CTACCCTCCCGGGCCCCAGG CCCAGCTCCC CGAGGACCTC 120 CTGCCACGGA CCCTCAGCCA GCTCCAGCCAGACGAGCTCA GCCCTAAGGT GGACAGCAGT 180 GTGGAGAGAC ACCATCTGAT GGCAGCCCTCAGTGCCTATG CTGCCCAGAG GCCCCCAGCT 240 CCCCCTGGGA AGGGCAGCCT GGAGCCGCAGTACCTTCTGC GCGCCCCGTC CAGAATGCCC 300 AGGCCCTTGT TGTCGCCAGC CGTCCCCCAGAAGTGGCCTT CACCTCTGGG AGATCCTGAA 360 GACCCCCCCA GCACAGGGGA AGGAGCACGGATTCACACTC TCCTGAAGGA CCTGCAGAGG 420 CAGCCGGCTG AGGCGAGGGG CCTGAGTGACCTGGAGCTGG ACAGCATGGC CGAGCTGATG 480 GCTGGCCTGA TGCAAGGCAT GGACCACAGAGGAGCTCTAG GCGGCCCTGG GAAAGCGGCC 540 CTGGGAGAGT CTGGAGAACA GGCGGATGGCCCCAAGGCCG CCCTCCGTGG GGAAAGCTTT 600 CCAGATGACG GAGTTCAGGA CGACGATGACAGACTTTACC AAGAGGTCCA TCGTCTGAGT 660 GCCACACTCG GGGGCCTCCT GCAGGACCACGGGTCTCGAC TCTCGCCTGG AGCCCTCCCC 720 TTTGCAAAGC CCCTCAAAAT GGAGAGGAAGAAATCCGAGC GCCCTGAGGC TTCCCTGTCT 780 TCAGAAGAGG AGACTGCCGG AGTGGAGAACGTCAAGAGCC AGACGTATTC CAAAACCTGC 840 TGGGGCAGCA GCCGCATTCG GAGCCCGGGGCAGGCGCGTT TGGGGAGCTC CAAACCAGAT 900 GCCTGGGCCC TCGGAGGAGG AGCAGAGCCTTCCAGCGGGT GCTCAGGAGG CCCTCGGCGA 960 CGGCTGCAAT TGGAAGTCAA GCCTTCCGAGGAAGAGGCAC GGTGCTACAT CGTGACAGAC 1020 AGAGACCCCC TGCGCCCCGA GGAAGGAAGGCAGCTGGTGG AGGACGTCGC CCGCCTCCTG 1080 CAGATGCCCA GCAGCACATT CGCCGACGTGGAGGTTCTCG GACCAGCAGT GACCTTCAAA 1140 GTGGGCGCCA ATGTCCAGAA CGTGACCACTGCGGATGTGG AGAAGGCCAC AGTTGACAAC 1200 AAAGACAAAC TGGAGGAAAC CTCTGGACTGAAAATTCTTC AAACCGGAGT CGGGTCGAAA 1260 AGCAAACTCA AGTTCCTGCC TCCTCAGGCGGAGCAAGAAG ACTCAACCAA GTTCATCGCG 1320 CTCACCCTGG TCTCCCTCGC CTGCATCCTGGGCGTCCTCC TGGCCTCTGG CCTCATCTAC 1380 TGCCTACGCC ATAGCTCTCA GCACAGGCTGAAGGAGAAGC TCTCGGGACT AGGGCGCGAC 1440 CCAGGTGCAG ATGCCACCGC CGCCTACCAGGAGCTGTGCC GCCAGCGTAT GGCCACGCGG 1500 CCACCAGACC GGCCCGAGGG CCCGCACACATCCCGCATCA GCAGCGTCTC GTCCCAGTTC 1560 AGCGACGGGC CGATGCCCAG CCCCTCCGCACGCAGCAGCG CCTCGTCCTG GTCCGAGGAG 1620 CCCGTGCAGT CCAACATGGA CATCTCCACCGGCCACATGA TCCTGTCCTA CATGGAGGAC 1680 CACCTGAAGA ACAAGAACCG GCTGGAGAAGGAGTGGGAGG CGCTGTGTGC CTACCAGGCG 1740 GAGCCCAACA GCTCACTTGT GGCCCAGAAGGAGGAGAATG TGCCCAAGAA CCGCTCCCTG 1800 GCCGTGCTGA CCTATGACCA CTCCCGGGTCCTACTGAAGG CGGAGAACAG CCACAGCCAC 1860 TCGGACTACA TCAACGCCAG CCCCATCATGGATCACGACC CGAGGAACCC CGCGTACATC 1920 GCCACCCAGG GACCGCTGCC CGCCACCGTGGCCGACTTTT GGCAGATGGT GTGGGAGAGC 1980 GGCTGCGTGG TGATCGTCAT GCTGACACCCCTCACAGAGA ACGGCGTCCG GCAGTGCTAC 2040 CACTACTGGC CAGATGAAGG CTCCAACCTCTACCACATCT ATGAGGTGAA CCTGGTCTCC 2100 GAGCACATCT GGTGCGAGGA CTTTCTGGTGAGGAGCTTCT ATCTGAAGAA CCTGCAGACC 2160 AACGAGACGC GCACCGTGAC CCAGTTCCACTTCCTGAGTT GGTATGACCG AGGAGTCCCC 2220 TCCTCCTCAA GATCCCTCCT GGACTTCCGCAGAAAAGTAA ACAAGTGCTA CAGGGGCCGT 2280 TCTTGTCCAA TAATTGTTCA TTGCAGTGACGGTGCAGGCC GGAGCGGCAC CTACGTCCTG 2340 ATCGACATGG TTCTCAACAA GATGGCCAAAGGTGCTAAAG AGATTGATAT CGCAGCAACC 2400 CTGGAGCACT TGAGGGACCA GAGACCCGGCATGGTCCAGA CGAAGGAGCA GTTTGAGTTC 2460 GCGCTGACAG CCGTGGCTGA AGAGGTGAATGCCATCCTCA AGGCCCTTCC CCAGTGAGCA 2520 GCGGCCTCGG GGCCTCGGGG GAGCCCCCACCCCCCGGATG TCGTCAGGAA TCGTGATCTG 2580 ACTTTAATTG TGTGTCTTCT ATTATAACTGCATAGTAATA GGGCCCTTAG CTCTCCCGTA 2640 GTCAGCGCAG TTTAGCAGTT AAGCAGTTAAAATGTGTATT TTTGTTTAAT CCAACAATAA 2700 TAAAGAGAGA TTTGTGGAAA AATCCCAAAAAAAAAA 2736 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 738 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 24: ACGTGGCAGG ATGACTATAC TCAGTATGTGATGGACCAGG AACTTGCAGA CCTCCCCAAA 60 ACCTACCTGA GGCATCCTGA AGCGTCCGGCCCAGCCAGGC CCTCAAAACA CAGCATTGGC 120 AGTGAGAGGA GGTACAGTCG GGAGGGCGGCGCTGCCCTGG CCAAGGCCTT CCGACGCCAC 180 CTGCCCTTCC TGGAGGCCCT GTCCCAGGCCCCAGCTTCAG ACGCGCTCGC CAGGACCCGG 240 ATGGCGCAGG ACAGACCCCG TGCTGAGGGTGACGACCGCT TCTCCAAGAG CATCCTGACC 300 TATGTGGCCC ACACGTCTGT GCTGACCTACCCTCCCGGGC CCCAGGCCCA GCTCCCCGAG 360 GACCTCCTGC CACGGACCCT CAGCCAGCTCCAGCCAGACG AGCTCAGCCC TAAGGTGGAC 420 AGCAGTGTGG AGAGACACCA TCTGATGGCAGCCCTCAGTG CCTATGCTGC CCAGAGGCCC 480 CCAGCTCCCC CTGGGAAGGG CAGCCTGGAGCCGCAGTACC TTCTGCGCGC CCCGTCCAGA 540 ATGCCCAGGC CCTTGTTGTC GCCAGCCGTCCCCCAGAAGT GGCCTTCACC TCTGGGAGAT 600 CCTGAAGACC CCCCCAGCAC AGGGGAAGGAGCACGGATTC ACACTCTCCT GAAGGACCTG 660 CAGAGGCAGC CGGCTGAGGC GAGGGGCCTGAGTGACCTGG AGCTGGACAG CATGGCCGAG 720 CTGATGGCTG GCCTGATG 738 (2)INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:932 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO: 25: GCCGGCAGCT CCCGGGGCGC CTGGGCTGCC TACTCGAGGA GGGCCTCTGCGGAGCGTCCG 60 AGGCCTGTGT GAACGATGGA GTGTTTGGAA GGTGCCAGAA GGTTCCGGCAATGGACTTTT 120 ACCGCTACGA GGTGTCGCCC GTGGCCCTGC AGCGCCTGCG CGTGGCTTTGCAGAAACTCT 180 CCGGCACAGG TTTCACGTGG CAGGATGACT ATACTCAGTA TGTGATGGACCAGGAACTTG 240 CAGACCTCCC CAAAACCTAC CTGAGGCATC CTGAAGCGTC CGGCCCAGCCAGGCCCTCAA 300 AACACAGCAT TGGCAGTGAG AGGAGGTACA GTCGGGAGGG CGGCGCTGCCCTGGCCAAGG 360 CCTTCCGACG CCACCTGCCC TTCCTGGAGG CCCTGTCCCA GGCCCCAGCTTCAGACGCGC 420 TCGCCAGGAC CCGGATGGCG CAGGACAGAC CCCGTGCTGA GGGTGACGACCGCTTCTCCA 480 AGAGCATCCT GACCTATGTG GCCCACACGT CTGTGCTGAC CTACCCTCCCGGGCCCCAGG 540 CCCAGCTCCC CGAGGACCTC CTGCCACGGA CCCTCAGCCA GCTCCAGCCAGACGAGCTCA 600 GCCCTAAGGT GGACAGCAGT GTGGAGAGAC ACCATCTGAT GGCAGCCCTCAGTGCCTATG 660 CTGCCCAGAG GCCCCCAGCT CCCCCTGGGA AGGGCAGCCT GGAGCCGCAGTACCTTCTGC 720 GCGCCCCGTC CAGAATGCCC AGGCCCTTGT TGTCGCCAGC CGTCCCCCAGAAGTGGCCTT 780 CACCTCTGGG AGATCCTGAA GACCCCCCCA GCACAGGGGA AGGAGCACGGATTCACACTC 840 TCCTGAAGGA CCTGCAGAGG CAGCCGGCTG AGGCGAGGGG CCTGAGTGACCTGGAGCTGG 900 ACAGCATGGC CGAGCTGATG GCTGGCCTGA TG 932 (2) INFORMATIONFOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 999 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTGTTGCTGC TACTGCTGCT GCTGCCGCCA CGCGTCCTGC CTGCCGCCCC CTCGTCCGTC 60CCCCACGGCC GGCAGCTCCC GGGGCGCCTG GGCTGCCTAC TCGAGGAGGG CCTCTGCGGA 120GCGTCCGAGG CCTGTGTGAA CGATGGAGTG TTTGGAAGGT GCCAGAAGGT TCCGGCAATG 180GACTTTTACC GCTACGAGGT GTCGCCCGTG GCCCTGCAGC GCCTGCGCGT GGCTTTGCAG 240AAACTCTCCG GCACAGGTTT CACGTGGCAG GATGACTATA CTCAGTATGT GATGGACCAG 300GAACTTGCAG ACCTCCCCAA AACCTACCTG AGGCATCCTG AAGCGTCCGG CCCAGCCAGG 360CCCTCAAAAC ACAGCATTGG CAGTGAGAGG AGGTACAGTC GGGAGGGCGG CGCTGCCCTG 420GCCAAGGCCT TCCGACGCCA CCTGCCCTTC CTGGAGGCCC TGTCCCAGGC CCCAGCTTCA 480GACGCGCTCG CCAGGACCCG GATGGCGCAG GACAGACCCC GTGCTGAGGG TGACGACCGC 540TTCTCCAAGA GCATCCTGAC CTATGTGGCC CACACGTCTG TGCTGACCTA CCCTCCCGGG 600CCCCAGGCCC AGCTCCCCGA GGACCTCCTG CCACGGACCC TCAGCCAGCT CCAGCCAGAC 660GAGCTCAGCC CTAAGGTGGA CAGCAGTGTG GAGAGACACC ATCTGATGGC AGCCCTCAGT 720GCCTATGCTG CCCAGAGGCC CCCAGCTCCC CCTGGGAAGG GCAGCCTGGA GCCGCAGTAC 780CTTCTGCGCG CCCCGTCCAG AATGCCCAGG CCCTTGTTGT CGCCAGCCGT CCCCCAGAAG 840TGGCCTTCAC CTCTGGGAGA TCCTGAAGAC CCCCCCAGCA CAGGGGAAGG AGCACGGATT 900CACACTCTCC TGAAGGACCT GCAGAGGCAG CCGGCTGAGG CGAGGGGCCT GAGTGACCTG 960GAGCTGGACA GCATGGCCGA GCTGATGGCT GGCCTGATG 999 (2) INFORMATION FOR SEQID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1011 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: GCGCTCCCGCTGCTGTTGCT GCTACTGCTG CTGCTGCCGC CACGCGTCCT GCCTGCCGCC 60 CCCTCGTCCGTCCCCCACGG CCGGCAGCTC CCGGGGCGCC TGGGCTGCCT ACTCGAGGAG 120 GGCCTCTGCGGAGCGTCCGA GGCCTGTGTG AACGATGGAG TGTTTGGAAG GTGCCAGAAG 180 GTTCCGGCAATGGACTTTTA CCGCTACGAG GTGTCGCCCG TGGCCCTGCA GCGCCTGCGC 240 GTGGCTTTGCAGAAACTCTC CGGCACAGGT TTCACGTGGC AGGATGACTA TACTCAGTAT 300 GTGATGGACCAGGAACTTGC AGACCTCCCC AAAACCTACC TGAGGCATCC TGAAGCGTCC 360 GGCCCAGCCAGGCCCTCAAA ACACAGCATT GGCAGTGAGA GGAGGTACAG TCGGGAGGGC 420 GGCGCTGCCCTGGCCAAGGC CTTCCGACGC CACCTGCCCT TCCTGGAGGC CCTGTCCCAG 480 GCCCCAGCTTCAGACGCGCT CGCCAGGACC CGGATGGCGC AGGACAGACC CCGTGCTGAG 540 GGTGACGACCGCTTCTCCAA GAGCATCCTG ACCTATGTGG CCCACACGTC TGTGCTGACC 600 TACCCTCCCGGGCCCCAGGC CCAGCTCCCC GAGGACCTCC TGCCACGGAC CCTCAGCCAG 660 CTCCAGCCAGACGAGCTCAG CCCTAAGGTG GACAGCAGTG TGGAGAGACA CCATCTGATG 720 GCAGCCCTCAGTGCCTATGC TGCCCAGAGG CCCCCAGCTC CCCCTGGGAA GGGCAGCCTG 780 GAGCCGCAGTACCTTCTGCG CGCCCCGTCC AGAATGCCCA GGCCCTTGTT GTCGCCAGCC 840 GTCCCCCAGAAGTGGCCTTC ACCTCTGGGA GATCCTGAAG ACCCCCCCAG CACAGGGGAA 900 GGAGCACGGATTCACACTCT CCTGAAGGAC CTGCAGAGGC AGCCGGCTGA GGCGAGGGGC 960 CTGAGTGACCTGGAGCTGGA CAGCATGGCC GAGCTGATGG CTGGCCTGAT G 1011 (2) INFORMATION FORSEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: ZC11654 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 28: CGGAATTCCT CTGTGGTCCA TGCCTTGC 28(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE:(B) CLONE: ZC11197 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: AAATTAATACGACTCACTAT AGGGAGACCG 30 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1210 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 30: AGTGCTGCTC ACTCTGGTGG CCCTGGCAGGTGTGGCTGGG CTGCTGGTGG CTCTGGCTGT 60 GGCTCTGTGT GTGCGGCAGC ATGCGCGGCAGCAAGACAAG GAGCGCCTGG CAGCCCTGGG 120 GCCTGAGGGG GCCCATGGTG ACACTACCTTTGAGTACCAG GACCTGTGCC GCCAGCACAT 180 GGCCACGAAG TCCTTGTTCA ACCGGGCAGAGGGTCCACCG GAGCCTTCAC GGGTGAGCAG 240 TGTGTCCTCC CAGTTCAGCG ACGCAGCCCAGGCCAGCCCC AGCTCCCACA GCAGCACCCC 300 GTCCTGGTGC GAGGAGCCGG CCCAAGCCAACATGGACATC TCCACGGGAC ACATGATTCT 360 GGCATACATG GAGGATCACC TGCGGAACCGGGACCGCCTT GCCAAGGAGT GGCAGGCCCT 420 CTGTGCCTAC CAAGCAGAGC CAAACACCTGTGCCACCGCG CAGGGGGAGG GCAACATCAA 480 AAAGAACCGG CATCCTGACT TCCTGCCCTATGACCATGCC CGCATAAAAC TGAAGGTGGA 540 GAGCAGCCCT TCTCGGAGCG ATTACATCAACGCCAGCCCC ATTATTGAGC ATGACCCTCG 600 GATGCCAGCC TACATAGCCA CGCAGGGCCCGCTGTCCCAT ACCATCGCAG ACTTCTGGCA 660 GATGGTGTGG GAGAGCGGCT GCACCGTCATCGTCATGCTG ACCCCGCTGG TGGAGGATGG 720 TGTCAAGCAG TGTGACCGCT ACTGGCCAGATGAGGGTGCC TCCCTCTACC ACGTATATGA 780 GGTGAACCTG GTGTCGGAGC ACATCTGGTGCGAGGACTTT CTGGTGCGGA GCTTCTACCT 840 GAAGAACGTG CAGACCCAGG AGACGCGCACGCTCACGCAG TTCCACTTCC TCAGCTGGCC 900 GGCAGAGGGC ACACCGGCCT CCACGCGGCCCCTGCTGGAC TTCCGCAGGA AGGTGAACAA 960 GTGCTACCGG GGCCGCTCCT GCCCCATCATCGTGCACTGC AGTGATGGTG CGGGGAGGAC 1020 CGGCACCTAC ATCCTCATCG ACATGGTCCTGAACCGCATG GCAAAAGGAG TGAAGGAGAT 1080 TGACATCGCT GCCACCCTGG AGCATGTCCGTGACCAGCGG CCTGGCCTTG TCCGCTCTAA 1140 GGACCAGTTT GAATTTGCCC TGACAGCCGTGGCGGAGGAA GTGAATGCCA TCCTCAAGGC 1200 CCTGCCCCAG 1210 (2) INFORMATIONFOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1263 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TCACACGTCT GTGCTGACCT ACCCTCCCGG GCCCCGGACC CAGCTCCACG AGGACCTCCT 60GCCACGGACC CTCGGCCAGC TCCAGCCAGA TGAGCTCAGC CCTAAGGTGG ACAGTGGTGT 120GGACAGACAC CATCTGATGG CGGCCCTCAG TGCCTATGCT GCCCAGAGGC CCCCAGCTCC 180CCCCGGGGAG GGCAGCCTGG AGCCACAGTA CCTTCTGCGT GCACCCTCAA GAATGCCCAG 240GCCTTTGCTG GCACCAGCCG CCCCCCAGAA GTGGCCTTCA CCTCTGGGAG ATTCCGAAGA 300CCCCTCTAGC ACAGGCGATG GAGCACGGAT TCATACCCTC CTGAAGGACC TGCAGAGGCA 360GCCGGCTGAG GTGAGGGGCC TGAGTGGCCT GGAGCTGGAC GGCATGGCTG AGCTGATGGC 420TGGCCTGATG CAAGGCGTGG ACCATGGAGT AGCTCGAGGC AGCCCTGGGA GAGCGGCCCT 480GGGAGAGTCT GGAGAACAGG CGGATGGCCC CAAGGCCACC CTCCGTGGAG ACAGCTTTCC 540AGATGACGGA GTGCAGGACG ACGATGATAG ACTTTACCAA GAGGTCCATC GTCTGAGTGC 600CACACTCGGG GGCCTCCTGC AGGACCACGG GTCTCGACTC TTACCTGGAG CCCTCCCCTT 660TGCAAGGCCC CTCGACATGG AGAGGAAGAA GTCCGAGCAC CCTGAGTCTT CCCTGTCTTC 720AGAAGAGGAG ACTGCCGGAG TGGAGAACGT CAAGAGCCAG ACGTATTCCA AAGATCTGCT 780GGGGCGGCAG CCGCATTCGG AGCCCGGGGC CGCTGCGTTT GGGGAGCTCC AAAACCAGAT 840GCCTGGGCCC TCGAAGGAGG AGCAGAGCCT TCCAGCGGGT GCTCAGGAGG CCCTCAGCGA 900CGGCCTGCAA TTGGAGGTCC AGCCTTCCGA GGAAGAGGCG CGGGGCTACA TCGTGACAGA 960CGGAGACCCC CTGCGCCCCG AGGAAGGAAG GCGGCTGGTG GAGGACGTCG CCCGCCTCCT 1020GCAGGTGCCC AGCAGCGCGT TCGCTGACGT GGAGGTTCTC GGACCAGCAG TGACCTTCAA 1080AGTGAGCGCC AATGTCCAAA ACGTGACCAC TGAGGATGTG GAGAAGGCCA CAGTTGACAA 1140CAAAGACAAA CTGGAGGAAA CCTCTGGACT GAAAATTCTT CAAACCGGAG TCGGGTCGAA 1200AAGCAAACTC AAGTTCCTGC CTCCTCAGGC GGAGCAAGAA GACTCCACCA AGTTCATCGC 1260GCA 1263 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 758 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 32: GAATTCGGCT TAAGGCGACG GTGGACAACAAAGACAAACT GGAGGAAACC TCTGGACTGA 60 AAATTCTTCA AACCGGAGTC GGGTCGAAAAGCAAACTCAA GTTCCTGCCT CCTCAGGCGG 120 AGCAAGAAGA CTCCACCAAG TTCATCGCGCTCACCCTGGT CTCCCTCGCC TGCATCCTGG 180 GCGTCCTCCT GGCCTCTGGC CTCATCTACTGCCTCCGCCA TAGCTCTCAG CACAGGCTGA 240 AGGAGAAGCT CTCGGGACTA GGGGGCGACCCAGGTGCAGA TGCCACTGCC GCCTACCAGG 300 AGCTGTGCCG CCAGCGTATG GCCACGCGGCCACCAGACCG ACCTGAGGGC CCGCACACGT 360 CACGCATCAG CAGCGTCTCA TCCCAGTTCAGCGACGGGCC GATCCCCAGC CCCTCCGCAC 420 GCAGCAGCGC CTCATCCTGG TCCGAGGAGCCTGTGCAGTC CAACATGGAC ATCTCCACCG 480 GCCACATGAT CCTGTCCTAC ATGGAGGACCACCTGAAGAA CAAGAACCGG CTGGAGAAGG 540 AGTGGGAAGC GCTGTGCGCC TACCAGGCGGAGCCCAACAG CTCGTTCGTG GCCCAGAGGG 600 AGGAGAACGT GCCCAAGAAC CGCTCCCTGGCCGTGCTGAC CTATGACCAC TCCCGGGTCC 660 TGCTGAAGGC GGAGAACAGC CACAGCCACTCAGACTACAT CAACGCTAGC CCCATCATGG 720 ATCACGACCC GAGGAACCCC GCGTACAAAGCCGAATTC 758 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1150 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 33: AAGCTTCCAC CATGCGCCAT AGCTCTCAGCACAGGCTGAA AGAGAAGCTC TCGGGACTAG 60 GGGGCGACCC AGGTGCAGAT GCCACTGCCGCCTACCAGGA GCTGCGCCGC CAGCGTATGG 120 CCACGCGGCC ACCAGACCGA CCTGAGGGCCCGCACACGTC ACGCATCAGC AGCGTCTCAT 180 CCCAGTTCAG CGACGGGCCG ATCCCCAGCCCCTCCGCACG CAGCAGCGCC TCATCCTGGT 240 CCGAGGAGCC TGTGCAGTCC AACATGGACATCTCCACCGG CCACATGATC CTGTCCTACA 300 TGGAGGACCA CCTGAAGAAC AAGAACCGGCTGGAGAAGGA GTGGGAAGCG CTGTGCGCCT 360 ACCAGGCGGA GCCCAACAGC TCGTTCGTGGCCCAGAGGGA GGAGAACGTG CCCAAGAACC 420 GCTCCCTGGC CGTGCTGACC TATGACCACTCCCGGGTCCT GCTGAAGGCG GAGAACAGCC 480 ACAGCCACTC AGACTACATC AACGCTAGCCCCATCATGGA TCACGACCCG AGGAACCCCG 540 CGTACATCGC CACCCAGGGA CCGCTGCCCGCCACCGTGGC TGACCTTTGG CAGATGGTGT 600 GGGAGAGCGG CTGCGTGGTG ATCGTCATGCTGACACCCCT CGCGGAGAAC GGCGTCCGGC 660 AGTGCTACCA CTACTGGCCG GATGAAGGCTCCAATCTCTA CCACATCTAT GAGGTGAACC 720 TGGTCTCCGA GCACATCTGG TGTGAGGACTTCCTGGTGAG GAGCTTCTAT CTGAAGAACC 780 TGCAGACCAA CGAGACGCGC ACCGTGACGCAGTTCCACTT CCTGAGTTGG TATGACCGAG 840 GAGTCCCTTC CTCCTCAAGG TCCCTCCTGGACTTCCGCAG AAAAGTAAAC AAGTGCTACA 900 GGGGCCGTTC TTGTCCAATA ATTGTTCATTGCAGTGACGG TGCAGGCCGG AGCGGCACCT 960 ACGTCCTGAT CGACATGGTT CTCAACAAGACGGCCAAAGG TGCTAAAGAG ATTGATATCG 1020 CAGCGACCCT GGAGCACTTG AGGGACCAGAGACCCGGCAT GTCCAGACGA AGGAGCAGTT 1080 TGAGTTCGCG CTGACAGCCG TGGCTGAGGAGGTGAACGCC ATCCTCAAGG CCCTGCCCCA 1140 GTGAGAATTC 1150 (2) INFORMATIONFOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2328 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GAATTCGGCT TGAGGAACCC CGCGTACATC GCCACCCAGG GACCGCTGCC CGCCACCGTG 60GCTGACTTTT GGCAGATGGT GTGGGAGAGC GGCTGCGTGG TGATCGTCAT GCAGACACCC 120CTCGCGGAGA ACGGCGTCCG GCAGTGCTAC CACTACTGGC CGGATGAAGG CTCCAATCTC 180TACCACATCT ATGAGGTGAA CCTGGTCTCC GAGCACATCT GGTGTGAGGA CTTCCTGGTG 240AGGAGCTTCT ATCTGAAGAA CCTGCAGACC AACGAGACGC GCACCGTGAC GCAGTTCCAC 300TTCCTGAGTT GGTTTGACCG AGGAGTCCCT TCCTCCTCAA GGTCCCTCCT GGACTTCCGC 360AGAAAAGTAA ACAAGTGCTA CAGGGGCCGT TCTTGTCCAA TAATTGTTCA TTGCAGTGAC 420GGTGCAGGCC GGAGCGGCAC CTACGTCCTG ATCGACATGG TTCTCAACAA GATGGCCAAA 480GGTGCTAAAG AGATTGATAT CGCAGCGACC CTGGAGCACT TGAGGGACCA GAGACCCGGC 540ATGGTCCAGA CGAAGGAGTA GTTTGAGTTC GCGCTGACAG CCGTGGCTGA GGAGGTGAAC 600GCCATCCTCA AGGCCCTTCC CCAGTGAGCG GCAGCCTCAG GGGCCTCAGG GGAGCCCCCA 660CCCCACGGAT GTTGTCAGGA ATCATGATCT GACTTTAATT GTGTGTCTTC TATTATAACT 720GCATAGTAAT AGGGCCCTTA GCTCTCCCGT AGTCAGCGCA GTTTAGCAGT TAAAAGTGTA 780TTTTTGTTTA ATCAAACAAT AATAAAGAGA GATTTGTGGA AAAATCCAGT TACGGGTGGA 840GGGGAATCGG TTCATCAATT TTCACTTGCT TAAAAAAAAT ACTTTTTCTT AAAGCACCCG 900TTCACCTTCT TGGTTGAAGT TGTGTTAACA ATGCAGTAGC CAGCACGTTC GAGGCGGTTT 960CCAGGAAGAG TGTGCTTGTC ATCTGCCACT TTCGGGAGGG TGGATCCACT GTGCAGGAGT 1020GGCCGGGGAA GCTGGCAGCA CTCAGTGAGG CCGCCCGGCA CACAAGGCAC GTTTGGCATT 1080TCTCTTTGAG AGAGTTTATC ATTGGGAGAA GCCGCGGGGA CAGAACTGAA CGTCCTGCAG 1140CTTCGGGGCA AGTGAGACAA TCACAGCTCC TCGCTGCGTC TCCATCAACA CTGCGCCGGG 1200TACCATGGAC GGCCCCGTCA GCCACACCTG TCAGCCCAAG CAGAGTGATT CAGGGGCTCC 1260CCGGGGGCAG GCACCTGTGC ACCCCATGAG TAGTGCCCAC TTGAGGCTGG CACTCCCCTG 1320ACCTCACCTT TGCAAAGTTA CAGATGCACC CCAACATTGA GATGTGTTTT TAATGTTAAA 1380ATATTGATTT CTACGTTATG AAAACAGATG CCCCCGTGAA TGCTTACCTG TGAGATAACC 1440ACAACCAGGA AGAACAAATC TGGGCATTGA GCAAGCTATG AGGGTCCCCG GGAGCACACG 1500AACCCTGCCA GGCCCCCGCT GGCTCCTCCA GGCACGTCCC GGACCTGTGG GGCCCCAGAG 1560AGGGGACATT TCCCTCCTGG GAGAGAAGGA GATCAGGGCA ACTCGGAGAG GGCTGCGAGC 1620ATTTCCCTCC CGGGAGAGGA GATCAGGGCG ACCTGCACGC ACTGCGTAGA GCCTGGAAGG 1680GAAGTGAGAA ACCAGCCGAC CGGCCCTGCC CCTCTTCCCG GGATCACTTA ATGAACCACG 1740TGTTTTGACA TCATGTAAAC CTAAGCACGT AGAGATGATT CGGATTTGAC AAAATAACAT 1800TTGAGTATCC GATTCGCCAT CACCCCCTAC CCCAGAAATA GGACAATTCA CTTCATTGAC 1860CAGGATGATC ACATGGAAGG CGGCGCAGAG GCAGCTGCGT GGGCTGCAGA TTTCCTGTGT 1920GGGGTTCAGC GTAGAAAACG CACCTCCATC CCGCCCTTCC CACAGCATTC CTCCATCTTA 1980GATAGATGGT ACTCTCCAAA GGCCCTACCA GAGGGAACAC GGCCTACTGA GCGGACAGAA 2040TGATGCCAAA ATATTGCTTA TGTCTCTACA TGGTATTGTA ATGAATATCT GCTTTAATAT 2100AGCTATCATT TCTTTTCCAA AATTACTTCT CTCTATCTGG AATTTAATTA ATCGAAATGA 2160ATTTATCTGA ATATAGGAAG CATATGCCTA CTTGTAATTT CTAACTCCTT ATGTTTGAAG 2220AGAAACCTCC GGTGTGAGAT ATACAAATAT ATTTAATTGT GTCATATTAA ACTTCTGATT 2280TCACCAAAAA AAAAAAAAAA AAAAAAAAAA AAAGCGGCCG CTGAATTC 2328

We claim:
 1. An isolated polynucleotide comprising a DNA segmentencoding a mammalian islet cell antigen polypeptide comprising an aminoacid sequence selected from the group consisting of: a) a polypeptide ofSEQ ID NO:16 from Leu, amino acid residue 636 to Gln, amino acid residue1012: b) a polypeptide of SEQ ID NO:22 from Leu, amino acid residue 442to Gln, amino acid residue 818; and c) allelic variants of (a) or (b)wherein the polypeptide forms an immune complex with an autoantibodyfrom a patient at risk of or predisposed to develop IDDM.
 2. An isolatedpolynucleotide according to claim 1, wherein said isolatedpolynucleotide encodes a mammalian islet cell antigen polypeptideselected from the group consisting of: a) a polypeptide of SEQ ID NO:16from Phe, amino acid residue 612 to Gln, amino acid residue 1012; b) apolypeptide of SEQ ID NO:22 from Phe, amino acid residue 418 to Gln,amino acid residue 818; and c) allelic variants of (a) or (b).
 3. Anisolated polynucleotide according to claim 1, wherein said isolatedpolynucleotide encodes a mammalian islet cell antigen polypeptideselected from the group consisting of: a) a polypeptide of SEQ ID NO:16from Ala, amino acid residue 1 to Gln, amino acid residue 1012; b) apolypeptide of SEQ ID NO:22 from His, amino acid residue 1 to Gln, aminoacid residue 818; and c) allelic variants of (a) or (b).
 4. An isolatedpolynucleotide according to claim 1, wherein said isolatedpolynucleotide is a DNA molecule selected from the group consisting of:a) a DNA molecule comprising the coding sequence of SEQ ID NO:15 fromnucleotide 1909 to nucleotide 3039; b) a DNA molecule comprising thecoding sequence of SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455;c) allelic variants of (a) or (b); and d) complements of polynucleotidemolecules that specifically hybridize to (a), (b) or (c).
 5. An isolatedpolynucleotide according to claim 1, wherein said isolatedpolynucleotide is a DNA molecule selected from the group consisting of:a) a DNA molecule comprising the coding sequence of SEQ ID NO:15 fromnucleotide 1837 to nucleotide 3039; b) a DNA molecule comprising thecoding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455; c)allelic variants of (a) or (b); and d) complements of polynucleotidemolecules that specifically hybridize to (a), (b) or (c).
 6. An isolatedpolynucleotide according to claim 1, wherein said isolatedpolynucleotide is a DNA molecule selected from the group consisting of:a) a DNA molecule comprising the coding sequence of SEQ ID NO:15 fromnucleotide 4 to nucleotide 3039; b) a DNA molecule comprising the codingsequence of SEQ ID NO:21 from nucleotide 1254 to nucleotide 2455; c)allelic variants of (a) or (b); and d) complements of polynucleotidemolecules that specifically hybridize to (a), (b) or (c).
 7. An isolatedpolynucleotide according to claim 1 which encodes a full lengthmammalian islet cell antigen polypeptide comprising the sequence of SEQID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue738.
 8. An isolated polynucleotide comprising a DNA segment encoding amammalian islet cell antigen polypeptide according to claim 1, whereinsaid mammalian islet cell antigen polypeptide is a primate islet cellantigen polypeptide.
 9. A DNA construct comprising a first DNA segmentencoding a human islet cell antigen polypeptide operably linked toadditional DNA segments required for the expression of said first DNAsegment, wherein said first DNA segment encodes a human islet cellantigen polypeptide comprising an amino acid sequence selected from thegroup consisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 toGln, amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acidresidue 418, to Gln, amino acid residue 818; c) a polypeptide of SEQ IDNO:22 from His, amino acid residue 1, to Gln, amino acid residue 818;and d) allelic variants of (a), (b) or (c) wherein said mammalian isletcell antigen polypeptide forms an immune complex with an autoantibodyfrom a patient at risk of or predisposed to develop IDDM.
 10. A DNAconstruct according to claim 9 herein said first DNA segment comprises anucleotide sequence selected from the group consisting of: a) a DNAmolecule comprising the coding sequence of SEQ. ID NO:21 from nucleotide1325 to nucleotide 2455; b) a DNA molecule comprising the codingsequence of SEQ ID NO:21 from nucleotide 1253 to nucleotide 2455; c) aDNA molecule comprising the coding sequence of SEQ ID NO:21 fromnucleotide 2 to nucleotide 2455; d) naturally occurring allelic variantsof (a), (b) or (c); and e) complements of polynucleotide molecules thatspecifically hybridize to (a), (b), (c) or (d).
 11. A DNA constructaccording to claim 9, wherein said first segment encodes a full lengthmammalian islet cell antigen comprising the amino acid sequence of SEQID NO:22 from Leu, amino acid residue 442 to Arg, amino acid residue738.
 12. A DNA construct comprising a first DNA segment encoding amammalian islet cell antigen according to claim 9, wherein saidmammalian islet cell antigen polypeptide is a primate islet cell antigenpolypeptide.
 13. A host cell containing a DNA construct comprising afirst DNA segment encoding a mammalian islet cell antigen polypeptideoperably linked to additional DNA segments required for the expressionof said first DNA segment, wherein said first DNA segment encodes ahuman islet cell antigen polypeptide comprising an amino acid sequenceselected from the group consisting of: a) SEQ ID NO:22 from Leu, aminoacid residue 442 to Gln, amino acid residue 818; b) SEQ ID NO:22 fromPhe, amino acid residue 418, to Gln, amino acid residue 818; c) apolypeptide of SEQ ID NO:22 from His, amino acid residue 1, to Gln,amino acid residue 818; and d) allelic variants of (a), (b) or (c)wherein said mammalian islet cell antigen polypeptide forms an immunecomplex with an autoantibody from a patient at risk of or predisposed todevelop IDDM.
 14. A host cell according to claim 13, wherein said firstDNA segment comprises a nucleotide sequence selected from the groupconsisting of: a) a DNA molecule comprising the coding sequence of SEQID NO:21 from nucleotide 1325 to nucleotide 2455; b) a DNA moleculecomprising the coding sequence of SEQ ID NO:21 from nucleotide 1253 tonucleotide 2455; c) a DNA molecule comprising the coding sequence of SEQID NO:21 from nucleotide 2 to nucleotide 2455; d) naturally occurringallelic variants of (a), (b) or (c); and e) complements ofpolynucleotide molecules that specifically hybridize to (a), (b), (c) or(d).
 15. A host cell according to claim 13, wherein said first DNAsegment encodes a full length mammalian islet cell antigen polypeptidecomprising the sequence of SEQ ID NO:22 from Leu, amino acid residue 442to Arg, amino acid residue
 738. 16. A host cell containing a DNAconstruct comprising a first DNA segment encoding a mammalian islet cellantigen polypeptide according to claim 13, wherein said mammalian isletcell antigen polypeptide is a primate islet cell antigen polypeptide.17. An isolated mammalian islet cell antigen polypeptide comprising anamino acid sequence selected from the group consisting of: a) SEQ IDNO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acidresidue 818; c) a polypeptide of SEQ ID NO:22 from His, amino acidresidue 1, to Gln, amino acid residue 818; and d) allelic variants of(a), (b) or (c) wherein said mammalian islet cell antigen polypeptideforms an immune complex with an autoantibody from a patient at risk ofor predisposed to develop IDDM.
 18. An isolated mammalian islet cellantigen polypeptide according to claim 17, wherein said isolatedmammalian islet cell antigen polypeptide is a full length mammalianislet cell antigen polypeptide comprising the sequence of SEQ ID NO:22from Leu, amino acid residue 442 to Arg, amino acid residue
 738. 19. Anisolated mammalian islet cell antigen polypeptide according to claim 17,wherein said mammalian islet cell antigen polypeptide is a primate isletcell antigen polypeptide.
 20. A method for producing a mammalian isletcell antigen polypeptide comprising the steps of: culturing a host cellcontaining a DNA construct comprising a first DNA segment operablylinked to additional DNA segments required for the expression of saidfirst DNA segment, wherein said first DNA segment encodes a human isletcell antigen polypeptide comprising an amino acid sequence selected fromthe group consisting of: a) SEQ ID NO:22 from Leu, amino acid residue442 to Gln, amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acidresidue 418, to Gln, amino acid residue 818; c) a polypeptide of SEQ IDNO:22 from His, amino acid residue 1, to Gln, amino acid residue 818;and d) allelic variants of (a), (b) or (c) wherein said mammalian isletcell antigen polypeptide forms an immune complex with an autoantibodyfrom a patient at risk of or predisposed to develop IDDM; and isolatingsaid mammalian islet cell antigen polypeptide.
 21. A method forproducing a mammalian islet cell antigen polypeptide according to claim20, wherein said first DNA segment comprises a nucleotide sequenceselected from the group consisting of: a) a DNA molecule comprising thecoding sequence of SEQ ID NO:21 from nucleotide 1325 to nucleotide 2455;b) a DNA molecule comprising the coding sequence of SEQ ID NO:21 fromnucleotide 1253 to nucleotide 2455; c) a DNA molecule comprising thecoding sequence of SEQ ID NO:21 from nucleotide 2 to nucleotide 2455; d)naturally occurring allelic variants of (a), (b) or (c); and e)complements of polynucleotide molecules that specifically hybridize to(a), (b), (c) or (d).
 22. A method for producing a mammalian islet cellantigen polypeptide according to claim 20, wherein said first DNAsegment encodes a full length mammalian islet cell antigen polypeptidecomprising the amino acid sequence of SEQ ID NO:22 from Leu, amino acidresidue 442 to Arg, amino acid residue
 738. 23. A method for producing ahuman islet cell antigen polypeptide according to claim 20, wherein saidhost cell is a bacterial cell or a cultured human cell.
 24. A method fordetermining the presence of an autoantibody to a human islet cellantigen polypeptide in a biological sample comprising the steps of:contacting a biological sample with a human islet cell antigenpolypeptide comprising an amino acid sequence selected from the groupconsisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acid residue418, to Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22from His, amino acid residue 1, to Gln, amino acid residue 818; and d)allelic variants of (a), (b) or (c), under conditions conducive toimmune complex formation, and detecting the presence of immune complexformation between said human islet cell antigen polypeptide and saidautoantibody to a human islet cell antigen, thereby determining thepresence of an autoantibody to said human islet cell antigen in saidbiological sample.
 25. The method of determining the presence of anautoantibody to a human islet cell antigen polypeptide according toclaim 24, wherein said human islet cell antigen polypeptide isdetectably labeled.
 26. A method of determining the presence of anautoantibody to a human islet cell antigen polypeptide according toclaim 24, wherein said human islet cell antigen polypeptide is a fulllength human islet cell antigen polypeptide comprising the amino acidsequence of SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, aminoacid residue
 738. 27. A method for predicting the clinical course ofIDDM in a patient comprising: testing a biological sample from a patientfor the presence of human islet cell antigen polypeptide comprising anamino acid sequence selected from the group consisting of: a) SEQ IDNO:22 from Leu, amino acid residue 442 to Gln, amino acid residue 818;b) SEQ ID NO:22 from Phe, amino acid residue 418, to Gln, amino acidresidue 818; c) a polypeptide of SEQ ID NO:22 from His, amino acidresidue 1, to Gln, amino acid residue 818; and d) allelic variants of(a), (b) or (c), wherein said polypeptide forms an immune complex withan autoantibody from a patient at risk of or predisposed to develop IDDMand classifying said patient for clinical course of diabetes based onthe presence or absence of mammalian islet cell antigen polypeptides insaid sample.
 28. A method for predicting the clinical course of IDDMaccording to claim 27, wherein said patient is further tested for one ormore additional predictive markers associated with risk of or protectionfrom IDDM.
 29. A method for predicting the clinical course of IDDMaccording to claim 27, wherein said predictive marker is an autoantibodyto an antigen selected from the group consisting of GAD65, IA-2/ICA512,or insulin.
 30. A method for predicting the clinical course of IDDMaccording to claim 27, wherein said predictive marker is a genotypeselected from the group consisting of HLA DR and HLA DQ.
 31. A methodfor predicting the clinical course of IDDM according to claim 27,wherein said predictive marker is a polymorphic region in the 5′flanking region of a human insulin gene.
 32. A method of predicting theclinical course of IDDM according to claim 27, wherein said human isletcell antigen polypeptide is a full length human islet cell antigenpolypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu,amino acid residue 442 to Arg, amino acid residue
 738. 33. A method oftreating a patient to prevent an autoimmune response to a human isletcell antigen polypeptide, the method comprising inducing immunologicaltolerance in said patient by administering a human islet cell antigenpolypeptide comprising an amino acid sequence selected from the groupconsisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acid residue418, to Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22from His, amino acid residue 1, to Gln, amino acid residue 818; and d)allelic variants of (a), (b) or (c), that specifically binds a humanislet cell antigen receptor on immature or mature T or B lymphocytes.34. A method of treating a patient to prevent an autoimmune response toa human islet cell antigen polypeptide according to claim 33, whereinsaid human islet cell antigen polypeptide is a full length human isletcell antigen polypeptide comprising the amino acid sequence of SEQ IDNO:22 from Leu, amino acid residue 442 to Arg, amino acid residue 738.35. A probe which comprises an oligonucleotide of at least about 16nucleotides, wherein said oligonucleotide is at least 85% identical to asequence of the human islet cell antigen DNA sequence of SEQ ID NOs: 15or
 21. 36. An isolated antibody which specifically binds to a humanislet cell antigen polypeptide, wherein said human islet cell antigenpolypeptide comprising an amino acid sequence selected from the groupconsisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acid residue418, to Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22from His, amino acid residue 1, to Gln, amino acid residue 818; and d)allelic variants of (a), (b) or (c).
 37. An isolated antibody accordingto claim 36, wherein said isolated antibody is a monoclonal antibody.38. An isolated antibody according to claim 36, wherein said human isletcell antigen polypeptide is a full length human islet cell antigenpolypeptide comprising the amino acid sequence of SEQ ID NO:22 from Leu,amino acid residue 442 to Arg, amino acid residue
 738. 39. A hybridomawhich produces a monoclonal antibody which specifically binds to a humanislet cell antigen polypeptide, wherein said human islet cell antigenpolypeptide comprises an amino acid sequence selected from the groupconsisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acid residue418, to Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22from His, amino acid residue 1, to Gln, amino acid residue 818; and d)allelic variants of (a), (b) or (c).
 40. A hybridoma according to claim39, wherein said human islet cell antigen polypeptide is a full lengthhuman islet cell antigen polypeptide comprising the amino acid sequenceof SEQ ID NO:22 from Leu, amino acid residue 442 to Arg, amino acidresidue
 738. 41. A diagnostic kit for use in detecting an autoantibodyto pancreatic β-islet cells, comprising a container containing a humanislet cell antigen polypeptide wherein said human islet cell antigenpolypeptide comprises an amino acid sequence selected from the groupconsisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 to Gln,amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acid residue418, to Gln, amino acid residue 818; c) a polypeptide of SEQ ID NO:22from His, amino acid residue 1, to Gln, amino acid residue 818; and d)allelic variants of (a), (b) or (c), wherein said polypeptide forms animmune complex with an autoantibody from a patient at risk of orpredisposed to develop IDDM, and one or more containers containingadditional reagents.
 42. A pharmaceutical composition which comprises ahuman islet cell antigen polypeptide, wherein said human islet cellantigen polypeptide comprises an amino acid sequence selected from thegroup consisting of: a) SEQ ID NO:22 from Leu, amino acid residue 442 toGln, amino acid residue 818; b) SEQ ID NO:22 from Phe, amino acidresidue 418, to Gln, amino acid residue 818; c) a polypeptide of SEQ IDNO:22 from His, amino acid residue 1, to Gln, amino acid residue 818;and d) allelic variants of (a), (b) or (c), wherein said polypeptideforms an immune complex with an autoantibody from a patient at risk ofor predisposed to develop IDDM in combination with a pharmaceuticallyacceptable carrier or vehicle.
 43. A method for monitoring the diseasestate in a patient comprising: testing a biological sample from apatient for the presence of mammalian islet cell antigenpost-translationally modified polypeptides; determining theconcentration of said polypeptides; and correlating levels of saidpolypeptides in said sample with the disease state in a patient.
 44. Amethod for monitoring the disease state in a patient according to claim43 wherein said human islet cell antigen post-translationally modifiedpolypeptide comprise the sequence of SEQ ID NO:22 from His, amino acidresidue 1 to Glu, amino acid residue
 227. 45. A method for monitoringthe disease state in a patient according to claim 43 wherein saidbiological sample is plasma or serum.
 46. A method for monitoring thedisease state in a patient comprising: exposing T cells to islet cellantigen 1851 peptides; detecting T cell reactivity; and correlating Tcell reactivity with disease state.
 47. A method for monitoring thedisease state according to claim 46 wherein said T cells are fromperipheral blood mononuclear cells from a prediabetic patient.
 48. Amethod for monitoring the disease state according to claim 46 whereinsaid disease state is conversion from prediabetes to diabetes.